Methods for inhibiting muscle atrophy

ABSTRACT

In one aspect, the invention relates to methods for treating muscle atrophy by providing to an animal in need thereof an effective amount of a compound. The compound can modulate the expression levels of multiple mRNA of a muscle atrophy signature. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/051,246, filed Feb. 23, 2016, which is a divisional of U.S.application Ser. No. 14/124,582 (which has a 371(c) date of Mar. 28,2014), which is a national phase entry under 35 U.S.C. § 371 ofInternational Application No. PCT/US2012/041119, filed Jun. 6, 2012,which claims the benefit of U.S. Provisional Application No. 61/493,969,filed on Jun. 6, 2011. The entire contents of each of the priorapplications are incorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under T32 GM073610, 1R01AR059115-01 and HL007121 awarded by the National Institutes of Health,as well as support from grant IBX000976A awarded by the Department ofVeterans Affairs. The government has certain rights in the invention.

BACKGROUND

Skeletal muscle atrophy is characteristic of starvation and a commoneffect of aging. It is also a nearly universal consequence of severehuman illnesses, including cancer, chronic renal failure, congestiveheart failure, chronic respiratory disease, insulin deficiency, acutecritical illness, chronic infections such as HIV/AIDS, muscledenervation, and many other medical and surgical conditions that limitmuscle use. However, medical therapies to prevent or reverse skeletalmuscle atrophy in human patients do not exist. As a result, millions ofindividuals suffer sequelae of muscle atrophy, including weakness,falls, fractures, opportunistic respiratory infections, and loss ofindependence. The burden that skeletal muscle atrophy places onindividuals, their families, and society in general, is tremendous.

The pathogenesis of skeletal muscle atrophy is not well understood.Nevertheless, important advances have been made. For example, it hasbeen described previously that insulin/IGF1 signaling promotes musclehypertrophy and inhibits muscle atrophy, but is reduced byatrophy-inducing stresses such as fasting or muscle denervation (BodineS C, et al. (2001) Nat Cell Biol 3(11): 1014-1019; Sandri M, et al.(2004) Cell 117(3):399-4121; Stitt T N, et al. (2004) Mol Cell14(3):395-403; Hu Z, et al. (2009) The Journal of clinical investigation119(10):3059-3069; Dobrowolny G, et al. (2005) The Journal of cellbiology 168(2): 193-199; Kandarian S C & Jackman R W (2006) Muscle &nerve 33(2):155-165; Hirose M, et al. (2001) Metabolism: clinical andexperimental 50(2):216-222; Pallafacchina G, et al. (2002) Proceedingsof the National Academy of Sciences of the United States of America99(14):9213-9218). The hypertrophic and anti-atrophic effects ofinsulin/IGF1 signaling are mediated at least in part through increasedactivity of phosphoinositide 3-kinase (PI3K) and its downstreameffectors, including Akt and mammalian target of rapamycin complex 1(mTORC1) Sandri M (2008) Physiology (Bethesda) 23:160-170; Glass D J(2005) The international journal of biochemistry & cell biology 37(10):1974-1984).

Another important advance came from microarray studies of atrophyingrodent muscle (Lecker S H, et al. (2004) Faseb J 18(1):39-51; Sacheck JM, et al. (2007) Faseb J 21(1): 140-155; Jagoe R T, et al. Faseb J16(13): 1697-1712). Those studies showed that several seeminglydisparate atrophy-inducing stresses (including fasting, muscledenervation and severe systemic illness) generated many common changesin skeletal muscle mRNA expression. Some of those atrophy-associatedchanges promote muscle atrophy in mice; these include induction of themRNAs encoding atroginI/MAFbx and MuRF1 (two E3 ubiquitin ligases thatcatalyze proteolytic events), and repression of the mRNA encoding PGC-1α (a transcriptional co-activator that inhibits muscle atrophy) (SandriM, et al. (2006) Proceedings of the National Academy of Sciences of theUnited States of America 103(44): 16260-16265; Wenz T, et al.Proceedings of the National Academy of Sciences of the United States ofAmerica 106(48):20405-20410; Bodine S C, et al. (2001) Science (NewYork, N.Y. 294(5547): 1704-1708; Lagirand-Cantaloube J, et al. (2008)The EMBO journal 27(8): 1266-1276; Cohen S, et al. (2009) The Journal ofcell biology 185(6): 1083-1095; Adams V, et al. (2008) Journal ofmolecular biology 384(1):48-59). However, the roles of many other mRNAsthat are increased or decreased in atrophying rodent muscle are not yetdefined. Data on the mechanisms of human muscle atrophy are even morelimited, although atrogin-1 and MuRF1 are likely to be involved (LegerB, et al. (2006) Faseb J 20(3):583-585; Doucet M, et al. (2007) Americanjournal of respiratory and critical care medicine 176(3):261-269; LevineS, et al. (2008) The New England journal of medicine 358(13):1327-1335).

Despite advances in understanding the physiology and pathophysiology ofmuscle atrophy, there is still a scarcity of compounds that are bothpotent, efficacious, and selective modulators of muscle growth and alsoeffective in the treatment of muscle atrophy associated and diseases inwhich the muscle atrophy or the need to increase muscle mass isinvolved. These needs and other needs are satisfied by the presentinvention.

SUMMARY

In accordance with the purpose(s) of the invention, as embodied andbroadly described herein, the invention, in one aspect, relates tocompounds useful in methods to treat muscle atrophy. The compounds canbe selected from a tacrine and analogs, naringenin and analogs,allantoin and analogs, conessine and analogs, tomatidine and analogs,ungerine/hippeastrine and analogs, and betulinic acid and analogs, or amixture thereof.

Tacrine and analogs can have the structure:

Naringenin and analogs can have the structure:

Allantoin and analogs can have the structure:

Conessine and analogs can have the structure:

Tomatidine and analogs can have the structure:

Ungerine/hippeastrine and analogs can have the structure:

Betulinic acid and analogs can have the structure:

The disclosed compounds can treat muscle atrophy when administered in aneffective amount to an animal, such as a mammal, fish or bird. Forexample, human.

Also disclosed in a method of lowering blood glucose in an animal byadministering ursolic acid or ursolic acid analogs, such as betulinincacid analogs, and narigenin analogs, such as naringenin, in an effectiveamount to an animal.

Also disclosed in a method of lowering blood glucose in an animal byadministering ungerine/hippeastrine analogs, such as hippeastrine, in aneffective amount to an animal.

The disclosed compounds can also promote muscle health, promote normalmuscle function, and/or promote healthy aging muscles by providing to asubject in need thereof an effective amount of a disclosed compound.

Also disclosed herein are pharmaceutical compositions comprisingcompounds used in the methods. Also disclosed herein are kits comprisingcompounds used in the methods.

In further aspects, In a further aspect, the invention relates tocompounds identified using muscle atrophy signature-1, muscle atrophysignature-2 or both muscle atrophy signatures. In still further aspects,the purpose(s) of the invention, as embodied and broadly describedherein, the invention, in one aspect, relates to compounds useful inmethods to modulate muscle health promote normal muscle function, and/orpromote healthy aging muscles, methods to inhibit muscle atrophy,methods to increase insulin/IGF-I signaling, methods to reduce body fat;methods to reduce blood glucose, methods to reduce blood triglycerides,methods to reduce blood cholesterol, methods to reduce obesity, methodsto reduce fatty liver disease, and methods to reduce diabetes, andpharmaceutical compositions comprising compounds used in the methods.

Disclosed are methods for treating muscle atrophy in a mammal, themethod comprising administering to the mammal an effective amount of acompound, wherein the compound: (a) down regulates multiple inducedmRNAs of a Muscle Atrophy Signature, compared to expression levels ofthe induced mRNAs of the Muscle Atrophy Signature in the same type ofthe muscle cell in the absence of the compound, and/or (b) up regulatesmultiple repressed mRNAs of the Muscle Atrophy Signature, compared toexpression levels of the repressed mRNAs of the Muscle Atrophy Signaturein the same type of the muscle cell in the absence of the compound,thereby inhibiting muscle atrophy in the mammal.

Also disclosed are methods for identifying a compound that inhibitsmuscle atrophy when administered in a effective amount to a animal inneed of treatment thereof, the method comprising the steps of: (i)selecting a candidate compound; (ii) determining the effect of thecandidate compound on a cell's expression levels of a plurality ofinduced mRNAs and/or repressed mRNAs of a Muscle Atrophy Signature,wherein the candidate compound is identified as suitable for muscleatrophy inhibition if: (a) more than one of the induced mRNAs of theMuscle Atrophy Signature are down regulated, compared to expressionlevels of the induced mRNAs of the Muscle Atrophy Signature in the sametype of cell in the absence of the candidate compound; and/or (b) morethan one of the repressed mRNAs of the Muscle Atrophy Signature are upregulated, compared to expression levels of the repressed mRNAs of theMuscle Atrophy Signature in the same type of cell in the absence of thecandidate compound. In one aspect, the method further comprisesadministering the candidate compound to an animal. The candidatecompound can be tacrine and analogs, naringenin and analogs, allantoinand analogs, conessine and analogs, tomatidine and analogs,ungerine/hippeastrine and analogs, and betulinic acid and analogs, or amixture thereof.

Also disclosed are methods for manufacturing a medicament associatedwith muscle atrophy or the need to promote muscle health, promote normalmuscle function, and/or promote healthy aging muscles comprisingcombining at least one disclosed compound or at least one disclosedproduct with a pharmaceutically acceptable carrier or diluent.

Also disclosed are uses of a disclosed compound or a disclosed productin the manufacture of a medicament for the treatment of a disorderassociated with muscle atrophy or the need to promote muscle health,promote normal muscle function, and/or promote healthy aging muscles.

While aspects of the present invention can be described and claimed in aparticular statutory class, such as the system statutory class, this isfor convenience only and one of skill in the art will understand thateach aspect of the present invention can be described and claimed in anystatutory class. Unless otherwise expressly stated, it is in no wayintended that any method or aspect set forth herein be construed asrequiring that its steps be performed in a specific order. Accordingly,where a method claim does not specifically state in the claims ordescriptions that the steps are to be limited to a specific order, it isno way intended that an order be inferred, in any respect. This holdsfor any possible non-express basis for interpretation, including mattersof logic with respect to arrangement of steps or operational flow, plainmeaning derived from grammatical organization or punctuation, or thenumber or type of aspects described in the specification.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects and together withthe description serve to explain the principles of the invention.

FIG. 1 shows a schematic overview of the discovery process leading to apharmacological compound that promotes skeletal muscle growth andinhibits skeletal muscle atrophy.

FIG. 2 shows human muscle atrophy signature-1.

FIG. 3 shows human muscle atrophy signature-2.

FIGS. 4A and 4B show representative data on the effect of fasting onskeletal muscle mRNA expression in healthy human adults.

FIG. 5 shows qPCR analysis of representative fasting-responsive mRNAsfrom human skeletal muscle.

FIGS. 6A-6H show representative data on the identification of ursolicacid as an inhibitor of fasting-induced skeletal muscle atrophy.

FIGS. 7A-7E show representative data on the identification of ursolicacid as an inhibitor of denervation-induced muscle atrophy.

FIGS. 8A-8E show representative data on ursolic acid-mediated inductionof muscle hypertrophy.

FIG. 9 shows representative data on the effect of ursolic acid on mouseskeletal muscle specific tetanic force.

FIGS. 10A-10K show representative data on the effect of ursolic acid onmuscle growth, atrophic gene expression, trophic gene expression, andskeletal muscle IGF-I signaling.

FIGS. 11A-11F show representative data on the effect of ursolic acid onskeletal muscle expression of IGF1 gene exons, adipose IGF1 mRNAexpression, and skeletal muscle insulin signaling.

FIGS. 12A-12J show representative data on the effect of ursolic acid onadiposity and plasma lipids.

FIGS. 13A-13F show representative data on the effect of ursolic acid onfood consumption, liver weight, kidney weight, and plasma ALT,bilirubin, and creatinine concentrations.

FIGS. 14A-14I show representative data on the effect of ursolic acid onweight gain, white adipose tissue weight, skeletal muscle weight, brownadipose tissue weight and energy expenditure in a mouse model of obesityand metabolic syndrome.

FIGS. 15A-15H show representative data on the effect of ursolic acid onobesity-related pre-diabetes, diabetes, fatty liver disease andhyperlipidemia in a mouse model of obesity and metabolic syndrome.

FIGS. 16A-16I show representative data that oleanolic acid and metformindo not reduce skeletal muscle atrophy.

FIGS. 17A and 17B show representative data that targeted inhibition ofPTP1B does not inhibit skeletal muscle atrophy.

FIGS. 18A and 18B show representative data on the effect of ursolic acidserum concentration on muscle mass and adiposity.

FIGS. 19A and 19B show that betulinic acid, like ursolic acid, reducesimmobilization-induced skeletal muscle atrophy. Mice were administeredvehicle (corn oil) or the indicated concentration of ursolic acid (A) orbetulinic acid (B) via intraperitoneal injection twice a day for twodays. One tibialis anterior (TA) muscle was immobilized with a surgicalstaple, leaving the contralateral mobile TA as an intrasubject control.Vehicle, or the same dose of ursolic acid or betulinic acid wasadministered via i.p. injection twice daily for six days beforecomparing weights of the immobile and mobile TAs. Data are means±SEMfrom 9-10 mice per condition. A, ursolic acid dose-responserelationship. B, betulinic acid dose-response relationship.

FIG. 20 shows that naringenin reduces immobilization-induced skeletalmuscle atrophy. Mice were administered vehicle (corn oil), ursolic acid(200 mg/kg), naringenin (200 mg/kg) or ursolic acid plus naringenin(both at 200 mg/kg) via intraperitoneal injection twice a day for twodays. One tibialis anterior (TA) muscle was immobilized with a surgicalstaple, leaving the contralateral mobile TA as an intrasubject control.Vehicle, or the same dose of ursolic acid and/or naringenin wasadministered via i.p. injection twice daily for six days beforecomparing weights of the immobile and mobile TAs. Data are means±SEMfrom 9-10 mice per condition.

FIGS. 21A-21F show that the combination of ursolic acid and naringeninnormalizes fasting blood glucose levels in a mouse model of glucoseintolerance, obesity and fatty liver disease. Mice were fed standardchow, high fat diet (HFD) plus the indicated concentrations ofnaringenin, or HFD containing 0.15% ursolic acid (UA) plus the indicatedconcentrations of naringenin for 5 weeks before measurement of fastingblood glucose (A), total body weight (B), fat mass by NMR (C), liverweight (D), grip strength (E) and skeletal muscle weight (bilateraltibialis anterior, gastrcocnemius, soleus, quadriceps and tricepsmuscle; F). Dashed line indicates levels in control mice that were fedstandard chow. Open symbols indicate levels in mice fed HFD containingthe indicated concentrations of naringenin. Closed symbols indicatelevels in mice fed HFD containing 0.15% UA plus the indicatedconcentrations of naringenin. Data are means±SEM from ≥12 mice percondition.

FIGS. 22A and 22B show that tomatidine reduces immobilization-inducedmuscle atrophy. Mice were administered vehicle (corn oil) or theindicated concentration of tomatidine via intraperitoneal injectiontwice a day for two days. One tibialis anterior (TA) muscle wasimmobilized with a surgical staple, leaving the contralateral mobile TAas an intrasubject control. Vehicle, or the same dose of tomatidine wasadministered via i.p. injection twice daily for six days beforecomparing weights of the immobile and mobile TAs. Data are means±SEMfrom 9-10 mice per condition. A, effects of 50, 100 and 200 mg/kgtomatidine. B, effects of 5, 15 and 50 mg/kg tomatidine.

FIGS. 23A and 23B show that tomatidine reduces fasting-induced muscleatrophy. Data are means±SEM from 9-12 mice per condition. Food waswithdrawn from mice, and then vehicle (corn oil), or the indicatedconcentrations of ursolic acid or tomatidine, were administered by i.p.injection. Twelve hours later, mice received another i.p. injection ofvehicle or the same dose of ursolic acid or tomatidine. Twelve hourslater, skeletal muscles (bilateral tibialis anterior, gastrcocnemius,soleus, quadriceps muscles) were harvested and weighed. A, comparison of200 mg/kg ursolic acid and 50 mg/kg tomatidine. B, effects of 5, 15 and50 mg/kg tomatidine.

FIG. 24 shows that allantoin, tacrine, ungerine, hippeastrine andconessine reduce fasting-induced muscle atrophy. Food was withdrawn frommice, and then vehicle or the indicated dose of ursolic acid,tomatidine, allantoin, tacrine, ungerine, hippeastrine or conessine wasadministered by i.p. injection. Twelve hours later, mice receivedanother i.p. injection of vehicle or the same dose of ursolic acid,tomatidine, allantoin, tacrine, ungerine, hippeastrine or conessine.Twelve hours later, skeletal muscles (bilateral tibialis anterior,gastrcocnemius and soleus muscles) were harvested and weighed. Data aremeans±SEM from ≥9 mice per condition and show the percent change inskeletal muscle weight relative to vehicle-treated animals in the sameexperiment. The vehicle for ursolic acid, tomatidine, ungerine,hippeastrine and conessine was corn oil. The vehicle for tacrine andallantoin was saline.

FIG. 25 shows that hippeastrine and conessine reduce fasting bloodglucose. Food was withdrawn from mice, and then vehicle or the indicateddose of hippeastrine or conessine was administered by i.p. injection.Twelve hours later, mice received another i.p. injection of vehicle orthe same dose of hippeastrine or conessine. Twelve hours later, bloodglucose was measured via tail vein. Data are means±SEM from ≥9 mice percondition.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or can be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DESCRIPTION

The present invention can be understood more readily by reference to thefollowing detailed description of the invention and the Examplesincluded therein.

Before the present compounds, compositions, articles, systems, devices,and/or methods are disclosed and described, it is to be understood thatthey are not limited to specific synthetic methods unless otherwisespecified, or to particular reagents unless otherwise specified, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only andis not intended to be limiting. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, example methods andmaterials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedherein can be different from the actual publication dates, which canrequire independent confirmation.

A. DEFINITIONS

As used herein, nomenclature for compounds, including organic compounds,can be given using common names, IUPAC, IUBMB, or CAS recommendationsfor nomenclature. When one or more stereochemical features are present,Cahn-Ingold-Prelog rules for stereochemistry can be employed todesignate stereochemical priority, E/Z specification, and the like. Oneof skill in the art can readily ascertain the structure of a compound ifgiven a name, either by systemic reduction of the compound structureusing naming conventions, or by commercially available software, such asCHEMDRAW™ (Cambridgesoft Corporation, U.S.A.).

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a functionalgroup,” “an alkyl,” or “a residue” includes mixtures of two or more suchfunctional groups, alkyls, or residues, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, a further aspect includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms a further aspect. It willbe further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

References in the specification and concluding claims to parts by weightof a particular element or component in a composition denotes the weightrelationship between the element or component and any other elements orcomponents in the composition or article for which a part by weight isexpressed. Thus, in a compound containing 2 parts by weight of componentX and 5 parts by weight component Y, X and Y are present at a weightratio of 2:5, and are present in such ratio regardless of whetheradditional components are contained in the compound.

A weight percent (wt. %) of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance can or can not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the term “muscle atrophy signature-1” refers to the setof mRNAs with an altered expression pattern associated with muscleatrophy. The mRNAs comprise mRNAs that are either induced or repressedduring the pathophysiology of muscle atrophy and which were identifiedusing the methods described herein. For clarity, muscle atrophysignature-1 comprise the induced and repressed mRNAs described in FIG.2.

As used herein, the term “muscle atrophy signature-2” refers to the setof mRNAs with an altered expression pattern associated with muscleatrophy. The mRNAs comprise mRNAs that are either induced or repressedduring the pathophysiology of muscle atrophy and which were identifiedusing the methods described herein. For clarity, muscle atrophysignature-2 comprise the induced and repressed mRNAs described in FIG.3.

As used herein, the term “muscle atrophy signature-3” refers to the setof mRNAs with an altered expression pattern associated with muscleatrophy. The mRNAs comprise mRNAs that are either induced or repressedduring the pathophysiology of muscle atrophy and which were identifiedusing the methods described herein. For clarity, muscle atrophysignature-3 comprise the induced and repressed mRNAs described inExample 23.

As used herein, the term “muscle atrophy signature-4” refers to the setof mRNAs with an altered expression pattern associated with muscleatrophy. The mRNAs comprise mRNAs that are either induced or repressedduring the pathophysiology of muscle atrophy and which were identifiedusing the methods described herein. For clarity, muscle atrophysignature-4 comprise the induced and repressed mRNAs described inExample 24.

As used herein, the term “subject” refers to the target ofadministration, e.g. an animal. Thus the subject of the herein disclosedmethods can be a vertebrate, such as a mammal, a fish, a bird, areptile, or an amphibian. Alternatively, the subject of the hereindisclosed methods can be a human, non-human primate, horse, pig, rabbit,dog, sheep, goat, cow, cat, guinea pig, fish, bird, or rodent. The termdoes not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered. In one aspect, the subject is a mammal. A patient refers to asubject afflicted with a disease or disorder. The term “patient”includes human and veterinary subjects. In some aspects of the disclosedmethods, the subject has been diagnosed with a need for treatment of oneor more muscle disorders prior to the administering step. In someaspects of the disclosed method, the subject has been diagnosed with aneed for promoting muscle health prior to the administering step. Insome aspects of the disclosed method, the subject has been diagnosedwith a need for promoting muscle health prior, promote normal musclefunction, and/or promote healthy aging muscles to the administeringstep.

As used herein, the term “treatment” refers to the medical management ofa patient with the intent to cure, ameliorate, stabilize, or prevent adisease, pathological condition, or disorder. This term includes the usefor astetic and self improvement purposes, for example, such usesinclude, but are not limited to, the administration of the disclosedcompound in nutraceuticals, medicinal food, energy bar, energy drink,supplements (such as multivitamins). This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder. In various aspects, the term covers anytreatment of a subject, including a mammal (e.g., a human), andincludes: (i) preventing the disease from occurring in a subject thatcan be predisposed to the disease but has not yet been diagnosed ashaving it; (ii) inhibiting the disease, i.e., arresting its development;or (iii) relieving the disease, i.e., causing regression of the disease.In one aspect, the subject is a mammal such as a primate, and, in afurther aspect, the subject is a human. The term “subject” also includesdomesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle,horses, pigs, sheep, goats, fish, bird, etc.), and laboratory animals(e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).

As used herein, the term “prevent” or “preventing” refers to precluding,averting, obviating, forestalling, stopping, or hindering something fromhappening, especially by advance action. It is understood that wherereduce, inhibit or prevent are used herein, unless specificallyindicated otherwise, the use of the other two words is also expresslydisclosed.

As used herein, the term “diagnosed” means having been subjected to aphysical examination by a person of skill, for example, a physician, andfound to have a condition that can be diagnosed or treated by thecompounds, compositions, or methods disclosed herein. For example,“diagnosed with a muscle atrophy disorder” means having been subjectedto a physical examination by a person of skill, for example, aphysician, and found to have a condition that can be diagnosed ortreated by a compound or composition that can promote muscle health,promote normal muscle function, and/or promote healthy aging muscles. Asa further example, “diagnosed with a need for promoting muscle health”refers to having been subjected to a physical examination by a person ofskill, for example, a physician, and found to have a conditioncharacterized by muscle atrophy or other disease wherein promotingmuscle health, promoting normal muscle function, and/or promotinghealthy aging muscles would be beneficial to the subject. Such adiagnosis can be in reference to a disorder, such as muscle atrophy, andthe like, as discussed herein.

As used herein, the phrase “identified to be in need of treatment for adisorder,” or the like, refers to selection of a subject based upon needfor treatment of the disorder. For example, a subject can be identifiedas having a need for treatment of a disorder (e.g., a disorder relatedto muscle atrophy) based upon an earlier diagnosis by a person of skilland thereafter subjected to treatment for the disorder. It iscontemplated that the identification can, in one aspect, be performed bya person different from the person making the diagnosis. It is alsocontemplated, in a further aspect, that the administration can beperformed by one who subsequently performed the administration.

As used herein, the terms “administering” and “administration” refer toany method of providing a pharmaceutical preparation to a subject. Suchmethods are well known to those skilled in the art and include, but arenot limited to, oral administration, transdermal administration,administration by inhalation, nasal administration, topicaladministration, intravaginal administration, ophthalmic administration,intraaural administration, intracerebral administration, rectaladministration, sublingual administration, buccal administration, andparenteral administration, including injectable such as intravenousadministration, intra-arterial administration, intramuscularadministration, and subcutaneous administration. Administration can becontinuous or intermittent. In various aspects, a preparation can beadministered therapeutically; that is, administered to treat an existingdisease or condition. In further various aspects, a preparation can beadministered prophylactically; that is, administered for prevention of adisease or condition.

The term “contacting” as used herein refers to bringing a disclosedcompound and a cell, target receptor, or other biological entitytogether in such a manner that the compound can affect the activity ofthe target (e.g., receptor, transcription factor, cell, etc.), eitherdirectly; i.e., by interacting with the target itself, or indirectly;i.e., by interacting with another molecule, co-factor, factor, orprotein on which the activity of the target is dependent.

As used herein, the terms “effective amount” and “amount effective”refer to an amount that is sufficient to achieve the desired result orto have an effect on an undesired condition. For example, a“therapeutically effective amount” refers to an amount that issufficient to achieve the desired therapeutic result or to have aneffect on undesired symptoms, but is generally insufficient to causeadverse side affects. The specific therapeutically effective dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the specific composition employed; the age, body weight, general health,sex and diet of the patient; the time of administration; the route ofadministration; the rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed and like factors well known in themedical arts. For example, it is well within the skill of the art tostart doses of a compound at levels lower than those required to achievethe desired therapeutic effect and to gradually increase the dosageuntil the desired effect is achieved. If desired, the effective dailydose can be divided into multiple doses for purposes of administration.Consequently, single dose compositions can contain such amounts orsubmultiples thereof to make up the daily dose. The dosage can beadjusted by the individual physician in the event of anycontraindications. Dosage can vary, and can be administered in one ormore dose administrations daily, for one or several days. Guidance canbe found in the literature for appropriate dosages for given classes ofpharmaceutical products. In further various aspects, a preparation canbe administered in a “prophylactically effective amount”; that is, anamount effective for prevention of a disease or condition.

As used herein, “EC₅₀,” is intended to refer to the concentration ordose of a substance (e.g., a compound or a drug) that is required for50% enhancement or activation of a biological process, or component of aprocess, including a protein, subunit, organelle, ribonucleoprotein,etc. EC₅₀ also refers to the concentration or dose of a substance thatis required for 50% enhancement or activation in vivo, as furtherdefined elsewhere herein. Alternatively, EC₅₀ can refer to theconcentration or dose of compound that provokes a response halfwaybetween the baseline and maximum response. The response can be measuredin a in vitro or in vivo system as is convenient and appropriate for thebiological response of interest. For example, the response can bemeasured in vitro using cultured muscle cells or in an ex vivo organculture system with isolated muscle fibers. Alternatively, the responsecan be measured in vivo using an appropriate research model such asrodent, including mice and rats. The mouse or rat can be an inbredstrain with phenotypic characteristics of interest such as obesity ordiabetes. As appropriate, the response can be measured in a transgenicor knockout mouse or rat wherein the a gene or genes has been introducedor knocked-out, as appropriate, to replicate a disease process.

As used herein, “IC₅₀,” is intended to refer to the concentration ordose of a substance (e.g., a compound or a drug) that is required for50% inhibition or diminuation of a biological process, or component of aprocess, including a protein, subunit, organelle, ribonucleoprotein,etc. IC₅₀ also refers to the concentration or dose of a substance thatis required for 50% inhibition or diminuation in vivo, as furtherdefined elsewhere herein. Alternatively, IC₅₀ also refers to the halfmaximal (50%) inhibitory concentration (IC) or inhibitory dose of asubstance. The response can be measured in a in vitro or in vivo systemas is convenient and appropriate for the biological response ofinterest. For example, the response can be measured in vitro usingcultured muscle cells or in an ex vivo organ culture system withisolated muscle fibers. Alternatively, the response can be measured invivo using an appropriate research model such as rodent, including miceand rats. The mouse or rat can be an inbred strain with phenotypiccharacteristics of interest such as obesity or diabetes. As appropriate,the response can be measured in a transgenic or knockout mouse or ratwherein the a gene or genes has been introduced or knocked-out, asappropriate, to replicate a disease process.

The term “pharmaceutically acceptable” describes a material that is notbiologically or otherwise undesirable, i.e., without causing anunacceptable level of undesirable biological effects or interacting in adeleterious manner.

As used herein, the term “derivative” refers to a compound having astructure derived from the structure of a parent compound (e.g., acompound disclosed herein) and whose structure is sufficiently similarto those disclosed herein and based upon that similarity, would beexpected by one skilled in the art to exhibit the same or similaractivities and utilities as the claimed compounds, or to induce, as aprecursor, the same or similar activities and utilities as the claimedcompounds. Exemplary derivatives include salts, esters, amides, salts ofesters or amides, and N-oxides of a parent compound.

As used herein, the term “pharmaceutically acceptable carrier” refers tosterile aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, as well as sterile powders for reconstitution into sterileinjectable solutions or dispersions just prior to use. Examples ofsuitable aqueous and nonaqueous carriers, diluents, solvents or vehiclesinclude water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol and the like), carboxymethylcellulose and suitablemixtures thereof, vegetable oils (such as olive oil) and injectableorganic esters such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants. These compositions can also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents and dispersingagents. Prevention of the action of microorganisms can be ensured by theinclusion of various antibacterial and antifungal agents such asparaben, chlorobutanol, phenol, sorbic acid and the like. It can also bedesirable to include isotonic agents such as sugars, sodium chloride andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the inclusion of agents, such as aluminummonostearate and gelatin, which delay absorption. Injectable depot formsare made by forming microencapsule matrices of the drug in biodegradablepolymers such as polylactide-polyglycolide, poly(orthoesters) andpoly(anhydrides). Depending upon the ratio of drug to polymer and thenature of the particular polymer employed, the rate of drug release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues. The injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable media just prior to use. Suitable inertcarriers can include sugars such as lactose. Desirably, at least 95% byweight of the particles of the active ingredient have an effectiveparticle size in the range of 0.01 to 10 micrometers.

A residue of a chemical species, as used in the specification andconcluding claims, refers to the moiety that is the resulting product ofthe chemical species in a particular reaction scheme or subsequentformulation or chemical product, regardless of whether the moiety isactually obtained from the chemical species. Thus, an ethylene glycolresidue in a polyester refers to one or more —OCH₂CH₂O— units in thepolyester, regardless of whether ethylene glycol was used to prepare thepolyester. Similarly, a sebacic acid residue in a polyester refers toone or more —CO(CH₂)₈CO— moieties in the polyester, regardless ofwhether the residue is obtained by reacting sebacic acid or an esterthereof to obtain the polyester.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described below. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms, such as nitrogen, canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. This disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds. Also, the terms“substitution” or “substituted with” include the implicit proviso thatsuch substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., a compound that does not spontaneouslyundergo transformation such as by rearrangement, cyclization,elimination, etc. It is also contemplated that, in certain aspects,unless expressly indicated to the contrary, individual substituents canbe further optionally substituted (i.e., further substituted orunsubstituted).

In defining various terms, “A¹,” “A²,” “A³,” and “A⁴” are used herein asgeneric symbols to represent various specific substituents. Thesesymbols can be any substituent, not limited to those disclosed herein,and when they are defined to be certain substituents in one instance,they can, in another instance, be defined as some other substituents.

The term “alkyl” as used herein is a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl,isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkylgroup can be cyclic or acyclic. The alkyl group can be branched orunbranched. The alkyl group can also be substituted or unsubstituted.For example, the alkyl group can be substituted with one or more groupsincluding, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether,halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein.A “lower alkyl” group is an alkyl group containing from one to six(e.g., from one to four) carbon atoms.

Throughout the specification “alkyl” is generally used to refer to bothunsubstituted alkyl groups and substituted alkyl groups; however,substituted alkyl groups are also specifically referred to herein byidentifying the specific substituent(s) on the alkyl group. For example,the term “halogenated alkyl” or “haloalkyl” specifically refers to analkyl group that is substituted with one or more halide, e.g., fluorine,chlorine, bromine, or iodine. The term “alkoxyalkyl” specifically refersto an alkyl group that is substituted with one or more alkoxy groups, asdescribed below. The term “alkylamino” specifically refers to an alkylgroup that is substituted with one or more amino groups, as describedbelow, and the like. When “alkyl” is used in one instance and a specificterm such as “alkylalcohol” is used in another, it is not meant to implythat the term “alkyl” does not also refer to specific terms such as“alkylalcohol” and the like.

This practice is also used for other groups described herein. That is,while a term such as “cycloalkyl” refers to both unsubstituted andsubstituted cycloalkyl moieties, the substituted moieties can, inaddition, be specifically identified herein; for example, a particularsubstituted cycloalkyl can be referred to as, e.g., an“alkylcycloalkyl.” Similarly, a substituted alkoxy can be specificallyreferred to as, e.g., a “halogenated alkoxy,” a particular substitutedalkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, thepractice of using a general term, such as “cycloalkyl,” and a specificterm, such as “alkylcycloalkyl,” is not meant to imply that the generalterm does not also include the specific term.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is atype of cycloalkyl group as defined above, and is included within themeaning of the term “cycloalkyl,” where at least one of the carbon atomsof the ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group andheterocycloalkyl group can be substituted or unsubstituted. Thecycloalkyl group and heterocycloalkyl group can be substituted with oneor more groups including, but not limited to, alkyl, cycloalkyl, alkoxy,amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol asdescribed herein.

The term “polyalkylene group” as used herein is a group having two ormore CH₂ groups linked to one another. The polyalkylene group can berepresented by the formula —(CH₂)_(a)—, where “a” is an integer of from2 to 500.

The terms “alkoxy” and “alkoxyl” as used herein to refer to an alkyl orcycloalkyl group bonded through an ether linkage; that is, an “alkoxy”group can be defined as —OA¹ where A¹ is alkyl or cycloalkyl as definedabove. “Alkoxy” also includes polymers of alkoxy groups as justdescribed; that is, an alkoxy can be a polyether such as —OA¹-OA² or—OA¹-(OA²)_(a)-OA³, where “a” is an integer of from 1 to 200 and A¹, A²,and A³ are alkyl and/or cycloalkyl groups.

The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon double bond. Asymmetric structures such as (A¹A²)C═C(A³A⁴)are intended to include both the E and Z isomers. This can be presumedin structural formulae herein wherein an asymmetric alkene is present,or it can be explicitly indicated by the bond symbol C═C. The alkenylgroup can be substituted with one or more groups including, but notlimited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, orthiol, as described herein.

The term “cycloalkenyl” as used herein is a non-aromatic carbon-basedring composed of at least three carbon atoms and containing at least onecarbon-carbon double bound, i.e., C═C. Examples of cycloalkenyl groupsinclude, but are not limited to, cyclopropenyl, cyclobutenyl,cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl,norbornenyl, and the like. The term “heterocycloalkenyl” is a type ofcycloalkenyl group as defined above, and is included within the meaningof the term “cycloalkenyl,” where at least one of the carbon atoms ofthe ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group andheterocycloalkenyl group can be substituted or unsubstituted. Thecycloalkenyl group and heterocycloalkenyl group can be substituted withone or more groups including, but not limited to, alkyl, cycloalkyl,alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone,azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon triple bond. The alkynyl group can be unsubstituted orsubstituted with one or more groups including, but not limited to,alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl,aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether,halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, asdescribed herein.

The term “cycloalkynyl” as used herein is a non-aromatic carbon-basedring composed of at least seven carbon atoms and containing at least onecarbon-carbon triple bound. Examples of cycloalkynyl groups include, butare not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and thelike. The term “heterocycloalkynyl” is a type of cycloalkenyl group asdefined above, and is included within the meaning of the term“cycloalkynyl,” where at least one of the carbon atoms of the ring isreplaced with a heteroatom such as, but not limited to, nitrogen,oxygen, sulfur, or phosphorus. The cycloalkynyl group andheterocycloalkynyl group can be substituted or unsubstituted. Thecycloalkynyl group and heterocycloalkynyl group can be substituted withone or more groups including, but not limited to, alkyl, cycloalkyl,alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone,azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

The term “aryl” as used herein is a group that contains any carbon-basedaromatic group including, but not limited to, benzene, naphthalene,phenyl, biphenyl, phenoxybenzene, and the like. The term “aryl” alsoincludes “heteroaryl,” which is defined as a group that contains anaromatic group that has at least one heteroatom incorporated within thering of the aromatic group. Examples of heteroatoms include, but are notlimited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term“non-heteroaryl,” which is also included in the term “aryl,” defines agroup that contains an aromatic group that does not contain aheteroatom. The aryl group can be substituted or unsubstituted. The arylgroup can be substituted with one or more groups including, but notlimited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiolas described herein. The term “biaryl” is a specific type of aryl groupand is included in the definition of “aryl.” Biaryl refers to two arylgroups that are bound together via a fused ring structure, as innaphthalene, or are attached via one or more carbon-carbon bonds, as inbiphenyl.

The term “aldehyde” as used herein is represented by the formula —C(O)H.Throughout this specification “C(O)” is a short hand notation for acarbonyl group, i.e., C═O.

The terms “amine” or “amino” as used herein are represented by theformula —NA¹A², where A¹ and A² can be, independently, hydrogen oralkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl,or heteroaryl group as described herein.

The term “alkylamino” as used herein is represented by the formula—NH(-alkyl) where alkyl is a described herein. Representative examplesinclude, but are not limited to, methylamino group, ethylamino group,propylamino group, isopropylamino group, butylamino group, isobutylaminogroup, (sec-butyl)amino group, (tert-butyl)amino group, pentylaminogroup, isopentylamino group, (tert-pentyl)amino group, hexylamino group,and the like.

The term “dialkylamino” as used herein is represented by the formula—N(-alkyl)₂ where alkyl is a described herein. Representative examplesinclude, but are not limited to, dimethylamino group, diethylaminogroup, dipropylamino group, diisopropylamino group, dibutylamino group,diisobutylamino group, di(sec-butyl)amino group, di(tert-butyl)aminogroup, dipentylamino group, diisopentylamino group, di(tert-pentyl)aminogroup, dihexylamino group, N-ethyl-N-methylamino group,N-methyl-N-propylamino group, N-ethyl-N-propylamino group and the like.

The term “carboxylic acid” as used herein is represented by the formula—C(O)OH.

The term “ester” as used herein is represented by the formula —OC(O)A¹or —C(O)OA¹, where A¹ can be alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.The term “polyester” as used herein is represented by the formula-(A¹O(O)C-A²-C(O)O)_(a)— or -(A¹O(O)C-A²-OC(O))_(a)—, where A¹ and A²can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and“a” is an integer from 1 to 500. “Polyester” is as the term used todescribe a group that is produced by the reaction between a compoundhaving at least two carboxylic acid groups with a compound having atleast two hydroxyl groups.

The term “ether” as used herein is represented by the formula A¹OA²,where A¹ and A² can be, independently, an alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group describedherein. The term “polyether” as used herein is represented by theformula -(A¹O-A²O)_(a)—, where A¹ and A² can be, independently, analkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl,or heteroaryl group described herein and “a” is an integer of from 1 to500. Examples of polyether groups include polyethylene oxide,polypropylene oxide, and polybutylene oxide.

The term “halide” as used herein refers to the halogens fluorine,chlorine, bromine, and iodine.

The term “heterocycle,” as used herein refers to single and multi-cyclicaromatic or non-aromatic ring systems in which at least one of the ringmembers is other than carbon. Heterocycle includes azetidine, dioxane,furan, imidazole, isothiazole, isoxazole, morpholine, oxazole, oxazole,including, 1,2,3-oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole,piperazine, piperidine, pyrazine, pyrazole, pyridazine, pyridine,pyrimidine, pyrrole, pyrrolidine, tetrahydrofuran, tetrahydropyran,tetrazine, including 1,2,4,5-tetrazine, tetrazole, including1,2,3,4-tetrazole and 1,2,4,5-tetrazole, thiadiazole, including,1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, thiazole,thiophene, triazine, including 1,3,5-triazine and 1,2,4-triazine,triazole, including, 1,2,3-triazole, 1,3,4-triazole, and the like.

The term “hydroxyl” as used herein is represented by the formula —OH.

The term “ketone” as used herein is represented by the formula A¹C(O)A²,where A¹ and A² can be, independently, an alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group asdescribed herein.

The term “azide” as used herein is represented by the formula —N₃.

The term “nitro” as used herein is represented by the formula —NO₂.

The term “nitrile” as used herein is represented by the formula —CN.

The term “silyl” as used herein is represented by the formula —SiA¹A²A³,where A¹, A², and A³ can be, independently, hydrogen or an alkyl,cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl,or heteroaryl group as described herein.

The term “sulfo-oxo” as used herein is represented by the formulas—S(O)A¹, —S(O)₂A¹, —OS(O)₂A¹, or —OS(O)₂OA¹, where A¹ can be hydrogen oran alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl,aryl, or heteroaryl group as described herein. Throughout thisspecification “S(O)” is a short hand notation for S═O. The term“sulfonyl” is used herein to refer to the sulfo-oxo group represented bythe formula —S(O)₂A¹, where A¹ can be hydrogen or an alkyl, cycloalkyl,alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl groupas described herein. The term “sulfone” as used herein is represented bythe formula A¹S(O)₂A², where A¹ and A² can be, independently, an alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, orheteroaryl group as described herein. The term “sulfoxide” as usedherein is represented by the formula A¹S(O)A², where A¹ and A² can be,independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, or heteroaryl group as described herein.

The term “thiol” as used herein is represented by the formula —SH.

“R¹,” “R²,” “R³,” “R^(n),” where n is an integer, as used herein can,independently, possess one or more of the groups listed above. Forexample, if R¹ is a straight chain alkyl group, one of the hydrogenatoms of the alkyl group can optionally be substituted with a hydroxylgroup, an alkoxy group, an alkyl group, a halide, and the like.Depending upon the groups that are selected, a first group can beincorporated within second group or, alternatively, the first group canbe pendant (i.e., attached) to the second group. For example, with thephrase “an alkyl group comprising an amino group,” the amino group canbe incorporated within the backbone of the alkyl group. Alternatively,the amino group can be attached to the backbone of the alkyl group. Thenature of the group(s) that is (are) selected will determine if thefirst group is embedded or attached to the second group.

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted,” whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. In is also contemplated that, in certain aspects,unless expressly indicated to the contrary, individual substituents canbe further optionally substituted (i.e., further substituted orunsubstituted).

The term “stable,” as used herein, refers to compounds that are notsubstantially altered when subjected to conditions to allow for theirproduction, detection, and, in certain aspects, their recovery,purification, and use for one or more of the purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(∘); —(CH₂)₀₋₄—OR^(∘); —O(CH₂)₁₋₄R^(∘),—O—(CH₂)₀₋₄C(O)OR^(∘); —(CH₂)₀₋₄CH(OR^(∘))₂; —(CH₂)₀₋₄SR^(∘);—(CH₂)₀₋₄Ph, which may be substituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Phwhich may be substituted with R^(∘); —CH═CHPh, which may be substitutedwith R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted withR^(∘); —NO₂; —CN; —N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘);—N(R^(∘))C(S)R^(∘); —(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘)₂; —(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄OC(O)R^(∘);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(∘); —(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR^(∘)₂; —C(S)NR^(∘) ₂; —C(S)SR^(∘); —SC(S)SR^(∘), —(CH₂)₀₋₄OC(O)NR^(∘) ₂;—C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘); —C(O)CH₂C(O)R^(∘);—C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘); —(CH₂)₀₋₄S(O)₂R^(∘);—(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋ ₄OS(O)₂R^(∘); —S(O)₂NR^(∘) ₂;—(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂; —N(R^(∘))S(O)₂R^(∘);—N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘); —P(O)R^(∘) ₂; —OP(O)R^(∘)₂; —OP(O)(OR^(∘))₂; SiR^(∘) ₃; —(C₁₋₄ straight or branchedalkylene)O—N(R^(∘))₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(R^(∘))₂, wherein each R^(∘) may be substituted asdefined below and is independently hydrogen, C₁-6 aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(∘), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(∘) (or the ring formed by takingtwo independent occurrences of R^(∘) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(haloR^(●)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●), —(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●), —(CH₂)₀₋₂NR^(●) ₂,—NO₂, —SiR^(●) ₃, —OSiR^(●) ₃, —C(O)SR^(●), —(C₁₋₄ straight or branchedalkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●) is unsubstituted orwhere preceded by “halo” is substituted only with one or more halogens,and is independently selected from C₁₋₄ aliphatic, —CH₂Ph, —O(CH₂)₀₋₁Ph,or a 5-6-membered saturated, partially unsaturated, or aryl ring having0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.Suitable divalent substituents on a saturated carbon atom of R^(∘)include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN,—C(O)OH, —C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein eachR^(●) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

The term “leaving group” refers to an atom (or a group of atoms) withelectron withdrawing ability that can be displaced as a stable species,taking with it the bonding electrons. Examples of suitable leavinggroups include halides and sulfonate esters, including, but not limitedto, triflate, mesylate, tosylate, and brosylate.

The terms “hydrolysable group” and “hydrolysable moiety” refer to afunctional group capable of undergoing hydrolysis, e.g., under basic oracidic conditions. Examples of hydrolysable residues include, withoutlimitatation, acid halides, activated carboxylic acids, and variousprotecting groups known in the art (see, for example, “Protective Groupsin Organic Synthesis,” T. W. Greene, P. G. M. Wuts, Wiley-Interscience,1999).

The term “organic residue” defines a carbon containing residue, i.e., aresidue comprising at least one carbon atom, and includes but is notlimited to the carbon-containing groups, residues, or radicals definedhereinabove. Organic residues can contain various heteroatoms, or bebonded to another molecule through a heteroatom, including oxygen,nitrogen, sulfur, phosphorus, or the like. Examples of organic residuesinclude but are not limited alkyl or substituted alkyls, alkoxy orsubstituted alkoxy, mono or di-substituted amino, amide groups, etc.Organic residues can preferably comprise 1 to 18 carbon atoms, 1 to 15,carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbonatoms, or 1 to 4 carbon atoms. In a further aspect, an organic residuecan comprise 2 to 18 carbon atoms, 2 to 15, carbon atoms, 2 to 12 carbonatoms, 2 to 8 carbon atoms, 2 to 4 carbon atoms, or 2 to 4 carbon atoms.

A very close synonym of the term “residue” is the term “radical,” whichas used in the specification and concluding claims, refers to afragment, group, or substructure of a molecule described herein,regardless of how the molecule is prepared. For example, a2,4-thiazolidinedione radical in a particular compound has the structure

regardless of whether thiazolidinedione is used to prepare the compound.In some embodiments the radical (for example an alkyl) can be furthermodified (i.e., substituted alkyl) by having bonded thereto one or more“substituent radicals.” The number of atoms in a given radical is notcritical to the present invention unless it is indicated to the contraryelsewhere herein.

“Organic radicals,” as the term is defined and used herein, contain oneor more carbon atoms. An organic radical can have, for example, 1-26carbon atoms, 1-18 carbon atoms, 1-12 carbon atoms, 1-8 carbon atoms,1-6 carbon atoms, or 1-4 carbon atoms. In a further aspect, an organicradical can have 2-26 carbon atoms, 2-18 carbon atoms, 2-12 carbonatoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Organicradicals often have hydrogen bound to at least some of the carbon atomsof the organic radical. One example, of an organic radical thatcomprises no inorganic atoms is a 5, 6, 7, 8-tetrahydro-2-naphthylradical. In some embodiments, an organic radical can contain 1-10inorganic heteroatoms bound thereto or therein, including halogens,oxygen, sulfur, nitrogen, phosphorus, and the like. Examples of organicradicals include but are not limited to an alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, mono-substituted amino,di-substituted amino, acyloxy, cyano, carboxy, carboalkoxy,alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide,substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl, thioalkyl,thiohaloalkyl, alkoxy, substituted alkoxy, haloalkyl, haloalkoxy, aryl,substituted aryl, heteroaryl, heterocyclic, or substituted heterocyclicradicals, wherein the terms are defined elsewhere herein. A fewnon-limiting examples of organic radicals that include heteroatomsinclude alkoxy radicals, trifluoromethoxy radicals, acetoxy radicals,dimethylamino radicals and the like.

“Inorganic radicals,” as the term is defined and used herein, contain nocarbon atoms and therefore comprise only atoms other than carbon.Inorganic radicals comprise bonded combinations of atoms selected fromhydrogen, nitrogen, oxygen, silicon, phosphorus, sulfur, selenium, andhalogens such as fluorine, chlorine, bromine, and iodine, which can bepresent individually or bonded together in their chemically stablecombinations. Inorganic radicals have 10 or fewer, or preferably one tosix or one to four inorganic atoms as listed above bonded together.Examples of inorganic radicals include, but not limited to, amino,hydroxy, halogens, nitro, thiol, sulfate, phosphate, and like commonlyknown inorganic radicals. The inorganic radicals do not have bondedtherein the metallic elements of the periodic table (such as the alkalimetals, alkaline earth metals, transition metals, lanthanide metals, oractinide metals), although such metal ions can sometimes serve as apharmaceutically acceptable cation for anionic inorganic radicals suchas a sulfate, phosphate, or like anionic inorganic radical. Inorganicradicals do not comprise metalloids elements such as boron, aluminum,gallium, germanium, arsenic, tin, lead, or tellurium, or the noble gaselements, unless otherwise specifically indicated elsewhere herein.

Compounds described herein can contain one or more double bonds and,thus, potentially give rise to cis/trans (E/Z) isomers, as well as otherconformational isomers. Unless stated to the contrary, the inventionincludes all such possible isomers, as well as mixtures of such isomers.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer and diastereomer, and a mixtureof isomers, such as a racemic or scalemic mixture. Compounds describedherein can contain one or more asymmetric centers and, thus, potentiallygive rise to diastereomers and optical isomers. Unless stated to thecontrary, the present invention includes all such possible diastereomersas well as their racemic mixtures, their substantially pure resolvedenantiomers, all possible geometric isomers, and pharmaceuticallyacceptable salts thereof. Mixtures of stereoisomers, as well as isolatedspecific stereoisomers, are also included. During the course of thesynthetic procedures used to prepare such compounds, or in usingracemization or epimerization procedures known to those skilled in theart, the products of such procedures can be a mixture of stereoisomers.

Many organic compounds exist in optically active forms having theability to rotate the plane of plane-polarized light. In describing anoptically active compound, the prefixes D and L or R and S are used todenote the absolute configuration of the molecule about its chiralcenter(s). The prefixes d and l or (+) and (−) are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or meaning that the compound is levorotatory. A compound prefixed with(+) or d is dextrorotatory. For a given chemical structure, thesecompounds, called stereoisomers, are identical except that they arenon-superimposable mirror images of one another. A specific stereoisomercan also be referred to as an enantiomer, and a mixture of such isomersis often called an enantiomeric mixture. A 50:50 mixture of enantiomersis referred to as a racemic mixture. Many of the compounds describedherein can have one or more chiral centers and therefore can exist indifferent enantiomeric forms. If desired, a chiral carbon can bedesignated with an asterisk (*). When bonds to the chiral carbon aredepicted as straight lines in the disclosed formulas, it is understoodthat both the (R) and (S) configurations of the chiral carbon, and henceboth enantiomers and mixtures thereof, are embraced within the formula.As is used in the art, when it is desired to specify the absoluteconfiguration about a chiral carbon, one of the bonds to the chiralcarbon can be depicted as a wedge (bonds to atoms above the plane) andthe other can be depicted as a series or wedge of short parallel linesis (bonds to atoms below the plane). The Cahn-Inglod-Prelog system canbe used to assign the (R) or (S) configuration to a chiral carbon.

Compounds described herein comprise atoms in both their natural isotopicabundance and in non-natural abundance. The disclosed compounds can beisotopically-labelled or isotopically-substituted compounds identical tothose described, but for the fact that one or more atoms are replaced byan atom having an atomic mass or mass number different from the atomicmass or mass number typically found in nature. Examples of isotopes thatcan be incorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine,such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F and ³⁶Cl,respectively. Compounds further comprise prodrugs thereof, andpharmaceutically acceptable salts of said compounds or of said prodrugswhich contain the aforementioned isotopes and/or other isotopes of otheratoms are within the scope of this invention. Certainisotopically-labelled compounds of the present invention, for examplethose into which radioactive isotopes such as ³H and ¹⁴C areincorporated, are useful in drug and/or substrate tissue distributionassays. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes areparticularly preferred for their ease of preparation and detectability.Further, substitution with heavier isotopes such as deuterium, i.e., ²H,can afford certain therapeutic advantages resulting from greatermetabolic stability, for example increased in vivo half-life or reduceddosage requirements and, hence, may be preferred in some circumstances.Isotopically labelled compounds of the present invention and prodrugsthereof can generally be prepared by carrying out the procedures below,by substituting a readily available isotopically labelled reagent for anon-isotopically labelled reagent.

The compounds described in the invention can be present as a solvate. Insome cases, the solvent used to prepare the solvate is an aqueoussolution, and the solvate is then often referred to as a hydrate. Thecompounds can be present as a hydrate, which can be obtained, forexample, by crystallization from a solvent or from aqueous solution. Inthis connection, one, two, three or any arbitrary number of solvate orwater molecules can combine with the compounds according to theinvention to form solvates and hydrates. Unless stated to the contrary,the invention includes all such possible solvates.

The term “co-crystal” means a physical association of two or moremolecules which owe their stability through non-covalent interaction.One or more components of this molecular complex provide a stableframework in the crystalline lattice. In certain instances, the guestmolecules are incorporated in the crystalline lattice as anhydrates orsolvates, see e.g. “Crystal Engineering of the Composition ofPharmaceutical Phases. Do Pharmaceutical Co-crystals Represent a NewPath to Improved Medicines?” Almarasson, O., et. al., The Royal Societyof Chemistry, 1889-1896, 2004. Examples of co-crystals includep-toluenesulfonic acid and benzenesulfonic acid.

It is also appreciated that certain compounds described herein can bepresent as an equilibrium of tautomers. For example, ketones with anα-hydrogen can exist in an equilibrium of the keto form and the enolform.

Likewise, amides with an N-hydrogen can exist in an equilibrium of theamide form and the imidic acid form. Unless stated to the contrary, theinvention includes all such possible tautomers.

It is known that chemical substances form solids which are present indifferent states of order which are termed polymorphic forms ormodifications. The different modifications of a polymorphic substancecan differ greatly in their physical properties. The compounds accordingto the invention can be present in different polymorphic forms, with itbeing possible for particular modifications to be metastable. Unlessstated to the contrary, the invention includes all such possiblepolymorphic forms.

In some aspects, a structure of a compound can be represented by aformula:

which is understood to be equivalent to a formula:

wherein n is typically an integer. That is, R^(n) is understood torepresent five independent substituents, R^(n(a)), R^(n(b)), R^(n(c)),R^(n(d)), R^(n(e)). By “independent substituents,” it is meant that eachR substituent can be independently defined. For example, if in oneinstance R^(n(a)) is halogen, then R^(n(b)) is not necessarily halogenin that instance.

Certain materials, compounds, compositions, and components disclosedherein can be obtained commercially or readily synthesized usingtechniques generally known to those of skill in the art. For example,the starting materials and reagents used in preparing the disclosedcompounds and compositions are either available from commercialsuppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), AcrosOrganics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), orSigma (St. Louis, Mo.) or are prepared by methods known to those skilledin the art following procedures set forth in references such as Fieserand Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wileyand Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition); and Larock'sComprehensive Organic Transformations (VCH Publishers Inc., 1989).

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Accordingly, where a method claim doesnot actually recite an order to be followed by its steps or it is nototherwise specifically stated in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatan order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including: matters of logic withrespect to arrangement of steps or operational flow; plain meaningderived from grammatical organization or punctuation; and the number ortype of embodiments described in the specification.

Disclosed are the components to be used to prepare the compositions ofthe invention as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds can not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular compound is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the compounds are discussed, specifically contemplated is eachand every combination and permutation of the compound and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the compositions of the invention. Thus, if there are avariety of additional steps that can be performed it is understood thateach of these additional steps can be performed with any specificembodiment or combination of embodiments of the methods of theinvention.

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures that can perform the same function that arerelated to the disclosed structures, and that these structures willtypically achieve the same result.

B. COMPOUNDS

In one aspect, the invention relates to compounds useful in methods toinhibit muscle atrophy by providing to a subject in need thereof aneffective amount of a compound or an analog thereof selected from amongthe compounds described herein, and pharmaceutical compositionscomprising compounds used in the methods. In a further aspect, theinvention relates to compounds identified using muscle atrophysignature-1, muscle atrophy signature-2, or both muscle atrophysignatures. In a further aspect, the invention relates to compoundsuseful in methods to modulate muscle health, methods to inhibit muscleatrophy, methods to increase insulin/IGF-I signaling, methods to reducebody fat; methods to reduce blood glucose, methods to reduce bloodtriglycerides, methods to reduce blood cholesterol, methods to reduceobesity, methods to reduce fatty liver disease, and methods to reducediabetes, and pharmaceutical compositions comprising compounds used inthe methods.

In one aspect, the compounds of the invention are useful in thetreatment of muscle disorders. In a further aspect, the muscle disordercan be skeletal muscle atrophy secondary to malnutrition, muscle disuse(secondary to voluntary or involuntary bedrest), neurologic disease(including multiple sclerosis, amyotrophic lateral sclerosis, spinalmuscular atrophy, critical illness neuropathy, spinal cord injury orperipheral nerve injury), orthopedic injury, casting, and otherpost-surgical forms of limb immobilization, chronic disease (includingcancer, congestive heart failure, chronic pulmonary disease, chronicrenal failure, chronic liver disease, diabetes mellitus, Cushingsyndrome, growth hormone deficiency, IGF-I deficiency, androgendeficiency, estrogen deficiency, and chronic infections such as HIV/AIDSor tuberculosis), burns, sepsis, other illnesses requiring mechanicalventiliation, drug-induced muscle disease (such as glucorticoid-inducedmyopathy and statin-induced myopathy), genetic diseases that primarilyaffect skeletal muscle (such as muscular dystrophy and myotonicdystrophy), autoimmune diseases that affect skeletal muscle (such aspolymyositis and dermatomyositis), spaceflight, or age-relatedsarcopenia.

It is contemplated that each disclosed derivative can be optionallyfurther substituted. It is also contemplated that any one or morederivative can be optionally omitted from the invention. It isunderstood that a disclosed compound can be provided by the disclosedmethods. It is also understood that the disclosed compounds can beemployed in the disclosed methods of using.

1. Tacrine and Analogs

In one aspect, the compound can be a tacrine analogs.

In one aspect, the tacrine analogs has a structure represented by aformula:

wherein R^(13a) and R^(13b) together comprise a cycle selected from:

wherein Q¹¹ is selected from N and CR^(13c);

wherein Q¹² is selected from N and CR^(13d);

wherein Q¹³ and Q¹⁴ are independently selected from CR^(13c)R^(13d), O,S, and NR^(14c);

wherein Q¹⁵ is selected from CR^(13c)R^(13d), O, S, and NR^(14c);

wherein Q¹⁶ is selected from N and CR^(13c);

wherein Q¹⁷ and Q¹⁸ are independently selected from CR^(13c)R^(13d), O,S, and NR^(14c);

wherein R¹¹ and R¹² are independently selected from H and C1-C6 alkyl;

wherein R^(13c), R^(13d), R^(13e), and R^(13f) are independentlyselected from H, C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro,amino, cyano, NHCOR¹⁵, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independentlyare substituted with 0, 1, 2, or 3 substituents selected from halogen,hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl,C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;

wherein each R^(14c) is independently selected from H and C1-C6 alkyl;

wherein R^(14a) and R^(14b) together comprise a cycle selected from:

wherein each of Q¹⁹, Q²⁰, Q²¹, Q²², Q²³, Q²⁴, and Q²⁵ are independentlyselected from CR^(17a)R^(17b), O, S, and NR¹⁸;

wherein R^(16a) and R^(16a) are independently selected from H, C1-C6alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR¹⁵, C1-C6monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, andC2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9heteroaryl, and C2-C9 heterocyclyl are independently are substitutedwith 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano,amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;

wherein R^(17a) and R^(17b) are independently selected from H, C1-C6alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR¹⁵, C1-C6monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, andC2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9heteroaryl, and C2-C9 heterocyclyl are independently are substitutedwith 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano,amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;

wherein each R¹⁸ is independently selected from H and C1-C6 alkyl;

wherein each R¹⁵ is independently selected from H and C1-C6 alkyl;

wherein each n is independently selected from 0, 1, and 2;

wherein m is selected from 1 and 2; and

wherein p is selected from 1, 2 and 3; or

a stereoisomer, tautomer, solvate, or pharmaceutically acceptable saltthereof.

In one aspect, compound (A) has the structure represented by theformula:

wherein R¹² is selected from H and C1-C6 alkyl;

wherein R^(13c), R^(13d), R^(13e), and R^(13f) are independentlyselected from H, C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro,amino, cyano, NHCOR¹⁵, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independentlyare substituted with 0, 1, 2, or 3 substituents selected from halogen,hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl,C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;

wherein Q¹⁹ and Q²⁰ are independently selected from CR^(17a)R^(17b), O,S, and NR¹⁸;

wherein R^(17a) and R^(17b) are independently selected from H, C1-C6alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR¹⁵, C1-C6monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, C2-C9heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl,and C2-C9 heterocyclyl are independently are substituted with 0, 1, 2,or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6alkylamino, and C1-C6 dialkylamino;

wherein each R¹⁸ is independently selected from H and C1-C6 alkyl; and

wherein n is selected from 0, 1, and 2.

In another aspect, R¹² is H; R^(13c), R^(13d), R^(13c), and R^(13f) areindependently selected from H, C1-C6 alkyl, C1-C6 alkoxy, halo,hydroxyl, nitro, and amino; Q¹⁹ and Q²⁰ are independently selected fromC R^(17a)R^(17b), O, S, and NR¹⁸; wherein R^(17a) and R^(17b) areindependently selected from H, C1-C6 alkyl, C1-C6 alkoxy, halo,hydroxyl, nitro, and amino; wherein each R¹⁸ is independently H; and nis selected from 0, 1, and 2.

In another aspect, R¹² is H; R^(13c), R^(13d), R^(13e), and R^(13f) areindependently selected from H, C1-C6 alkyl, C1-C6 alkoxy, halo,hydroxyl, nitro, and amino; Q¹⁹ and Q²⁰ are independently selected fromC R^(17a)R^(17b); wherein R^(17a) and R^(17b) are independently selectedfrom H, C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, and amino; andn is 1.

In another aspect, R¹² is H; R^(13c), R^(13d), R^(13e), and R^(13f) areindependently selected from H, C1-C6 alkyl, C1-C6 alkoxy, halo, andhydroxyl; Q¹⁹ and Q²⁰ are independently selected from CR^(17a)R^(17b);wherein R^(17a) and R^(17b) are independently H; and n is 1.

In another aspect, R¹² is H; R¹² is H; R^(13c), R^(13d), R^(13e), andR^(13f) are independently selected from H, C1-C6 alkyl, and halo; Q¹⁹and Q²⁰ are independently CR^(17a)R^(17b); wherein R^(17a) and R^(17b)are independently H; and wherein n is 1.

In another aspect, the formula is:]

2. Naringenin and Analog

In one aspect, the compound can be a naringenin analog.

In one aspect, the naringenin analog has a structure represented by aformula:

wherein each ----- represents a covalent bond selected from a single ordouble bond;

wherein R^(21a), R^(21b), R^(21c), R^(21d), and R^(21e) areindependently selected from H, OH, O-Glucosyl, halo, cyano, amino,nitro, nitroso, NHCOR¹⁵, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl,C1-C6 polyhaloalkyl, C1-C6 alkylamino, acyl, phenyl-C1-C6 alkoxy,benzyl-C1-C6 alkoxy, and C1-C6 dialkylamino;

wherein R²² is selected from H, OH, O— Glucosyl, halo, cyano, amino,nitro, nitroso, NHCOR¹⁵, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl,C1-C6 polyhaloalkyl, C1-C6 alkylamino, acyl phenyl-C1-C6 alkoxy,benzyl-C1-C6 alkoxy, and C1-C6 dialkylamino;

wherein R^(23a), R^(23b), R^(23c), and R^(23d) are independentlyselected from H, OH, O-Glucosyl halo, cyano, amino, nitro, nitroso,NHCOR¹⁵, C1-C20 alkyl, C1-C20 alkenyl, C1-C20 alkynyl, C1-C20 alkenynyl,C1-C20 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6alkylamino, acyl, phenyl-C1-C6 alkoxy, benzyl-C1-C6 alkoxy, and C1-C6dialkylamino;

wherein R¹⁵ is selected from H and C1-C6 alkyl;

wherein Z is selected from O and S; and

wherein Y is selected from O and S; or

a stereoisomer, tautomer, solvate, or pharmaceutically acceptable saltthereof;

wherein the compound does not have the structure:

In one aspect, ----- indicates a covalent single bond. In anotheraspect, ----- indicates a covalent double bond.

In another aspect, Z is O, and Y is O.

In another aspect, R^(21a), R^(21b), R^(21c), R^(21d), and R^(21e) areindependently selected from H and OH; wherein R²² is selected from H andOH; and wherein R^(23a), R^(23b), R^(23c), R^(23d), and R^(23e) areindependently selected from H and OH.

In another aspect, R^(21a), R^(21b), R^(21c), R^(21d), and R^(21e) areindependently selected from H, OH, O-Glucosyl, halo, cyano, amino,nitro, and nitroso.

In another aspect, R²² is H.

In another aspect, R^(21a), R^(21b), R^(21d), and R^(21e) are H, andR^(21c) is OH.

In another aspect, R^(23a) and R^(23c) are H, and R^(23b) and R^(23d)are OH.

In another aspect, R^(21a), R^(21b), R^(21d), and R^(21e) are H, R^(21c)is OH, R^(23a) and R^(23c) are H, and R^(23b) and R^(23d) are OH.

In another aspect, R^(21a), R^(21d), and R^(21e) are H, R^(21b) andR^(21c) are OH, R^(23a) and R^(23c) are H, and R^(23b) and R^(23d) areOH.

In another aspect, the compound has the structure:

3. Allantoin and Analogs

In one aspect, the compound can be a allantoin analog.

In one aspect, the allantoin analog has a structure represented by aformula:

wherein R^(31a) and R^(31b) are independently selected from H, C1-C6alkyl, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl,and C2-C9 heterocyclyl are independently are substituted with 0, 1, 2,or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6alkylamino, and C1-C6 dialkylamino;

wherein R^(32a) and R^(32b) are independently selected from H, C1-C6alkyl, OCl(OH)₄Al₂, OAl(OH)₂, C1-C6 alkoxy, halo, hydroxyl, nitro,amino, cyano, NHCOR¹⁵, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9heteroaryl, and C2-C9 heterocyclyl or taken together to form a doublebond selected from ═O and ═S, wherein C6-C10 aryl, C3-C10 cycloalkyl,C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently aresubstituted with 0, 1, 2, or 3 substituents selected from halogen,hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl,C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;

wherein R^(32c) and R^(32d) are independently selected from H, C1-C6alkyl, OCl(OH)₄Al₂, OAl(OH)₂, C1-C6 alkoxy, halo, hydroxyl, nitro,amino, cyano, NHCOR¹⁵, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9heteroaryl, and C2-C9 heterocyclyl or taken together to form a doublebond selected from ═O and ═S, wherein C6-C10 aryl, C3-C10 cycloalkyl,C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently aresubstituted with 0, 1, 2, or 3 substituents selected from halogen,hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl,C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;

wherein R^(33a) and R^(33b) are independently selected from H,NR^(34a)CONR^(34b)R^(34c), C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl,nitro, amino, cyano, NHCOR¹⁵, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl,C1-C6 alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl,C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independentlyare substituted with 0, 1, 2, or 3 substituents selected from halogen,hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl,C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino; and

wherein R^(34a)R^(34b) and R^(34c) are independently selected from H,C1-C6 alkyl, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl,and C2-C9 heterocyclyl are independently are substituted with 0, 1, 2,or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6alkylamino, and C1-C6 dialkylamino; or

a stereoisomer, tautomer, solvate, or pharmaceutically acceptable saltthereof.

In one aspect, R^(31a) and R^(31b) are H.

In another aspect, R^(32a) and R^(32b) are taken together to form ═O.

In another aspect, R^(32c) and R^(32d) are taken together to form ═O.

In another aspect, R^(32a) and R^(32b) are taken together to form ═O,and R^(32c) and R^(32d) are taken together to form ═O.

In another aspect, R^(31a) is H, R^(31b) is H, R^(32a), and R^(32b) aretaken together to form ═O, and R^(32c) and R^(32d) are taken together toform ═O.

In another aspect, R^(31a) is H, R^(31b) is H, R^(32a) and R^(32b) aretaken together to form ═O, R^(32c) and R^(32d) are taken together toform ═O, and one of R^(33a) and R^(33b) is NR^(34a)CONR^(34b)R^(34c) andthe other one of R^(33a) and R^(33b) is H.

In another aspect, one of R^(33a) and R^(33b) isNR^(34a)CONR^(34b)R^(34c) and the other one of R^(33a) and R^(33b) is H.

In another aspect, one of R^(32a) and R^(32b) is OCl(OH)₄Al₂ and theother one of R^(32a) and R^(32b) is H.

In another aspect, one of R^(32c) and R^(32d) is OCl(OH)₄Al₂ and theother one of R^(32c) and R^(32b) is H.

In another aspect, one of R^(32a) and R^(32b) is OAl(OH)₂ and the otherone of R^(32a) and R^(32b) is H.

In another aspect, one of R^(32c) and R^(32d) is OAl(OH)₂ and the otherone of R^(32c) and R^(32d) is H.

In another aspect, the compound has the structure:

4. Conessine and Analogs

In one aspect, the compound can be a conessine analog.

In one aspect, the conessine analog has a structure represented by aformula:

wherein each ----- represents a covalent bond independently selectedfrom a single or double bond, wherein valency is satisfied;

wherein R⁴¹ is selected from NR^(48a)R^(48b), ═O, ═S, C1-C6 alkoxy andhydroxyl;

wherein R^(48a) and R^(48b) are independently selected from H, C1-C6alkyl, C1-C6 heteroalkyl, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independentlysubstituted with 0, 1, 2, or 3 substituents selected from halogen,hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl,C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;

wherein R⁴² is selected from H, C1-C6 alkoxy and hydroxyl;

wherein R⁴³ is selected from H and C1-C6 alkyl;

wherein R^(44a) and R^(44b) are independently selected from areindependently selected from H, hydroxyl, and C1-C6 alkoxy;

wherein R^(47a) and R^(47b) are independently selected from areindependently selected from H, hydroxyl, and C1-C6 alkoxy;

wherein R^(45a) and R^(45b) together comprise a cycle selected from:

wherein R^(49a) is selected from H and C1-C6 alkyl; and

wherein R^(49b) and R^(49c) are independently selected from H and C1-C6alkyl, or taken together to form ═O; or

a stereoisomer, tautomer, solvate, or pharmaceutically acceptable saltthereof.

In one aspect, R^(47a) and R^(47b) are independently selected from H,hydroxyl, and C1-C6 alkoxy.

In another aspect, R^(44a) and R^(44b) are independently selected fromH, hydroxyl, and C1-C6 alkoxy.

In another aspect, R⁴² is H.

In another aspect, R^(47a) and R^(47b) are selected from H, hydroxyl,and C1-C6 alkoxy; R^(44a) and R^(44b) are H.

In another aspect, R⁴¹ is selected from NR^(48a)R^(48b) and ═O, whereinR^(48a) and R^(48b) are independently selected from H and C1-C6 alkyl.

In another aspect, R⁴³ is C1 alkyl.

In another aspect, the formula has the structure:

In another aspect, the formula has the structure:

In another aspect, the formula has the structure:

In another aspect, the formula has the structure:

In another aspect, the formula has the structure:

5. Tomatidine and Analogs

In one aspect, the compound can be a tomatidine analog.

In one aspect, the tomatidine analog has a structure represented by aformula:

wherein R⁵¹ is selected from H, C1-C6 alkyl, COR⁵³, C1-C6 alkylamino,C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl,and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0,1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino,C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl,C1-C6 alkylamino, and C1-C6 dialkylamino;

wherein R⁵³ is selected from C1-C6 alkyl, C1-C6 monohaloalkyl, C1-C6polyhaloalkyl, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, andC2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0,1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino,C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl,C1-C6 alkylamino, and C1-C6 dialkylamino;

wherein Z⁵¹ is selected from O, S, and NR⁵⁴;

wherein R⁵⁴ is selected from H, C1-C6 alkyl, COR⁵⁵, C1-C6 alkylamino,C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl,and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0,1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino,C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl,C1-C6 alkylamino, and C1-C6 dialkylamino;

wherein R⁵⁵ is selected from C1-C6 alkyl, C1-C6 monohaloalkyl, C1-C6polyhaloalkyl, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, andC2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0,1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino,C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl,C1-C6 alkylamino, and C1-C6 dialkylamino; or

a stereoisomer, tautomer, solvate, or pharmaceutically acceptable saltthereof.

In one aspect, R⁵¹ is selected from H, C1-C6 alkyl and COR³, wherein R⁵³is C1-C6 alkyl.

In another aspect, R⁵¹ is H.

In another aspect, Z⁵¹ is NR⁵⁴. In another aspect, Z⁵¹ is NR⁵⁴, whereinR⁵⁴ is selected from H, C1-C6 alkyl, and COR⁵⁵, wherein R⁵⁵ is C1-C6alkyl.

In another aspect, R⁵¹ is selected from H, C1-C6 alkyl and COR⁵³,wherein R⁵³ is C1-C6 alkyl; and Z⁵¹ is NR⁵⁴, wherein R⁵⁴ is selectedfrom H, C1-C6 alkyl, and COR⁵⁵, wherein R⁵⁵ is C1-C6 alkyl.

In another aspect, R⁵¹ and R⁵⁴ are identical.

In another aspect, the structure is represented by the formula:

In another aspect, the structure is represented by the formula:

In another aspect, the formula has the structure:

6. Ungerine/Hippeastrine and Analogs

In one aspect, the compound can be a ungerine/hippeastrine analog.

In one aspect, the ungerine/hippeastrine has a structure represented bya formula:

wherein R^(61a) and R^(61b) are independently selected from H, C1-C6alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR¹⁵, C1-C6monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, andC2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0,1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino,C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl,C1-C6 alkylamino, and C1-C6 dialkylamino;

wherein R^(62a) and R^(62b) are independently selected from H, C1-C6alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR¹⁵, C1-C6monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, andC2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0,1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino,C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl,C1-C6 alkylamino, and C1-C6 dialkylamino;

wherein R^(63a) and R^(63b) are independently selected from H, C1-C6alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR¹⁵, C1-C6monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, andC2-C9 heterocyclyl, or taken together to form a group selected from ═Oand ═S, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, andC2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl,C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6alkylamino, and C1-C6 dialkylamino;

wherein R^(64a) and R^(64b) are independently selected from H, OR⁶⁷,C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR¹⁵,C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, andC2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0,1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino,C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl,C1-C6 alkylamino, and C1-C6 dialkylamino;

wherein R⁶⁵ is selected from H, C1-C6 alkyl, C1-C6 alkoxy, C1-C6monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6dialkylamino, COR⁶⁶, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl,and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0,1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino,C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl,C1-C6 alkylamino, and C1-C6 dialkylamino;

wherein R⁶⁶ is selected from C1-C6 alkyl, C1-C6 monohaloalkyl, C1-C6polyhaloalkyl, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, andC2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0,1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino,C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl,C1-C6 alkylamino, and C1-C6 dialkylamino;

wherein R⁶⁷ is selected from C1-C6 alkyl, C1-C6 monohaloalkyl, C1-C6polyhaloalkyl, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, andC2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0,1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino,C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl,C1-C6 alkylamino, and C1-C6 dialkylamino;

wherein each R¹⁵ is independently selected from H and C1-C6 alkyl; or

a stereoisomer, tautomer, solvate, or pharmaceutically acceptable saltthereof,

wherein the compound is present in an effective amount.

In one aspect, the structure is represented by a formula:

In another aspect, the structure is represented by a formula:

In another aspect, R^(61a), R^(61b), R^(62a), and R^(62b) are H.

In another aspect, one of R^(63a) and R^(63b) is hydroxyl and the otherone of R^(63a) and R^(63b) is H.

In another aspect, R^(63a) and R^(63b) are taken together and form ═O.

In another aspect, one of R^(64a) and R^(64b) is hydroxyl or OR⁶⁷ andthe other one of R^(64a) and R^(64b) is H.

In another aspect, one of R^(64a) and R^(64b) is hydroxyl or OR⁶⁷ andthe other one of R^(64a) and R^(64b) is H, wherein R⁶⁷ is C1-C6 alkyl.

In another aspect, R⁶⁵ is C1-C6 alkyl.

In another aspect, the structure is represented by the formula:

In another aspect, the structure is represented by a formula:

In another aspect, the structure is represented by a formula:

In another aspect, the structure is represented by the formula:

In another aspect, the structure is represented by a formula:

7. Betulinic Acid and Analogs

In one aspect, the compound can be a betulinic acid derivative.

In one aspect, has a structure represented by a formula:

wherein ----- is an covalent bond selected from a single bond and adouble bond, wherein valency is satisfied, and R⁷⁰ is optionallypresent; wherein n is 0 or 1; wherein R⁷⁰, when present, is hydrogen;wherein R^(71a) is selected from C1-C6 alkyl and —C(O)ZR⁸²; whereinR^(71b) is selected from C1-C6 alkyl, or wherein R^(71a) and R^(71b) areoptionally covalently bonded and, together with the intermediate carbon,comprise an optionally substituted C3-C5 cycloalkyl or C2-C5heterocycloalkyl; wherein one of R^(72a) and R^(72b) is —Z⁷², and theother is hydrogen, or R^(72a) and R^(72b) together comprise ═O; whereineach of R^(73a) and R^(73b) is independently selected from hydrogen,hydroxyl, C1-C6 alkyl, and C1-C6 alkoxyl, provided that R^(73a) andR^(73b) are not simultaneously hydroxyl, wherein R^(73a) and R^(73b) areoptionally covalently bonded and, together with the intermediate carbon,comprise an optionally substituted C3-C5 cycloalkyl or C2-C5heterocycloalkyl; wherein each of R⁷⁴, R⁷⁵, and R⁷⁶ is independentlyselected from C1-C6 alkyl; wherein R⁷⁷ is selected from C1-C6 alkyl, and—C(O)Z⁷¹R⁸⁰; wherein R⁸⁰ is selected from hydrogen and C1-C6 alkyl;wherein R^(78a) and R^(78b) are independently selected from hydrogen andC1-C6 alkyl; wherein each of R^(79a) and R^(79b) is independentlyselected from hydrogen and C1-C6 alkyl, C2-C6 alkenyl, and C2-C6alkynyl, provided that R^(79a) and R^(79b) are not simultaneouslyhydrogen; or wherein R^(79a) and R^(79b) are covalently bonded and,along with the intermediate carbon, together comprise C3-C5 cycloalkylor C2-C5 heterocycloalkyl; wherein R⁸² is selected from hydrogen andC1-C6 alkyl; wherein Z⁷¹ and Z⁷² are independently selected from —OR⁸¹—and —NR⁸³—; wherein R⁸³ and R⁸³ are independently selected from hydrogenand C1-C4 alkyl; or, wherein Z⁷¹ and Z⁷² are independently N, R⁸⁴ andR⁸⁵ are covalently bonded and —NR⁸⁴R⁸⁵ comprises a moiety of theformula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—, or astereoisomer, tautomer, solvate, or pharmaceutically acceptable saltthereof.

In another aspect, the formula has the structure:

In another aspect, the formula has the structure:

In another aspect, the formula has the structure:

In one aspect, ----- is a single bond. In another aspect, ----- is adouble bond.

In one aspect, n is 0. In another aspect, n is 1.

In another aspect, R^(71a) is C1-C6 alkyl; R^(71b) is selected fromC1-C6 alkyl; one of R^(72a) is —Z⁷², and R^(72b) is hydrogen; R⁷⁴, R⁷⁵are independently selected from C1-C6 alkyl; wherein R^(79b) is selectedfrom C1-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl; Z⁷¹ is —O—; and Z⁷²is selected from —OR⁸ and —NR⁸³—; R⁸¹ and R⁸³ are independently selectedfrom hydrogen and C1-C4 alkyl.

In another aspect, R^(71a) is C1 alkyl; R^(71b) is C1 alkyl; R^(72a) is—Z⁷², and R^(72b) is hydrogen; R⁷⁴, R⁷⁵ are independently selected fromC1 alkyl; wherein R^(79b) is selected from C1-C6 alkyl, C2-C6 alkenyl,and C2-C6 alkynyl; Z⁷¹ is —O—; and Z⁷² is selected from —OR⁸¹ and—NR⁸³—; wherein R⁸¹ and R⁸³ are hydrogen.

In another aspect, R^(71a) is C1 alkyl; R^(71b) is C1 alkyl; R^(72a) is—Z⁷², and R^(72b) is hydrogen; R⁷⁴, R⁷⁵ are independently selected fromC1 alkyl; R^(79b) is C2-C6 alkenyl; Z⁷¹ is —O—; and Z⁷² is selected from—OR⁸¹ and —NR⁸³—; wherein R⁸¹ and R⁸³ are hydrogen.

8. Compounds Identified by Muscle Atrophy Signature-1 and Muscle AtrophySignature-2.

In various aspects, the invention relates to uses of one or morecompounds selected from tacrine analogs, naringenin analogs, allantoinanalogs, conessine analogs, tomatidine analogs, hippeastrine/ungerineanalogs and betulinic acid analogs.

a. Muscle Atrophy Signature-1

In one aspect, the disclosed compounds comprise compounds identifiedusing muscle atrophy signature-1. Such compounds include, but are notlimited to, allantoin; conessine; naringenin; tacrine; tomatidine or apharmaceutically acceptable salt, tautomer, stereoisomer, hydrate,solvate, or polymorph thereof. In a yet further aspect, the compound isan analog of one the preceding compounds as defined above.

b. Muscle Atrophy Signature-2

In a further aspect, the disclosed compounds comprise compoundsidentified using muscle atrophy signature-2. Such compounds include, butare not limited to, allantoin; betulinic acid; conessine; naringenin;tacrine; tomatidine or a pharmaceutically acceptable salt, tautomer,stereoisomer, hydrate, solvate, or polymorph thereof. In a yet furtheraspect, the compound is an analog of one the preceding compounds asdefined above.

c. Muscle Atrophy Signature-1 or Muscle Atrophy Signature-2

In a further aspect, the disclosed compounds comprise compoundsidentified using either muscle atrophy signature-1 or muscle atrophysignature-2. Such compounds include, but are not limited to, allantoin;betulinic acid; conessine; naringenin; tacrine; tomatidine or apharmaceutically acceptable salt, tautomer, stereoisomer, hydrate,solvate, or polymorph thereof. In a yet further aspect, the compound isan analog of one the preceding compounds as defined above.

d. Muscle Atrophy Signature-1 and Muscle Atrophy Signature-2

In a further aspect, the disclosed compounds comprise compoundsidentified using both muscle atrophy signature-1 and muscle atrophysignature-2, and is a compound associated with both muscle atrophysignatures. Such compounds include, but are not limited to, allantoin;conessine; naringenin; tacrine; tomatidine or a pharmaceuticallyacceptable salt, tautomer, stereoisomer, hydrate, solvate, or polymorphthereof. In a yet further aspect, the compound is an analog of one thepreceding compounds as defined above.

9. Inhibition of Muscle Atrophy

In one aspect, the disclosed compounds inhibit muscle atrophy. In afurther aspect, the disclosed compounds promoting muscle health,promoting normal muscle function, and/or promoting healthy agingmuscles. In a yet further aspect, the disclosed compounds inhibit ofmuscle atrophy and promote muscle health, promote normal musclefunction, and/or promote healthy aging muscles. In a further aspect, theinhibition of muscle atrophy is in an animal. In an even further aspect,the promoting muscle health, promoting normal muscle function, and/orpromoting healthy aging muscles is in an animal. In a still furtheraspect, the animal is a mammal, In a yet further aspect, the mammal is ahuman. In a further aspect, the mammal is a mouse. In a yet furtheraspect, the mammal is a rodent.

In a further aspect, the disclosed compounds inhibit muscle atrophy whenadministered at an oral dose of greater than about 5 mg per day in ahuman. In a further aspect, the disclosed compounds inhibit muscleatrophy when administered at an oral dose of greater than about 10 mgper day in a human. In a further aspect, the disclosed compounds inhibitmuscle atrophy when administered at an oral dose of greater than about25 mg per day in a human. In a further aspect, the disclosed compoundsinhibit muscle atrophy when administered at an oral dose of greater thanabout 50 mg per day in a human. In a further aspect, the disclosedcompounds inhibit muscle atrophy when administered at an oral dose ofgreater than about 75 mg per day in a human. In a further aspect, thedisclosed compounds inhibit muscle atrophy when administered at an oraldose of greater than about 100 mg per day in a human. In a furtheraspect, the disclosed compounds inhibit muscle atrophy when administeredat an oral dose of greater than about 150 mg per day in a human. In afurther aspect, the disclosed compounds inhibit muscle atrophy whenadministered at an oral dose of greater than about 200 mg per day in ahuman. In a further aspect, the disclosed compounds inhibit muscleatrophy when administered at an oral dose of greater than about 250 mgper day in a human. In a yet further aspect, the disclosed compoundsinhibit muscle atrophy when administered at an oral dose of greater thanabout 300 mg per day in a human. In a still further aspect, thedisclosed compounds inhibit muscle atrophy when administered at an oraldose of greater than about 400 mg per day in a human. In an even furtheraspect, the disclosed compounds inhibit muscle atrophy when administeredat an oral dose of greater than about 500 mg per day in a human. In afurther aspect, the disclosed compounds inhibit muscle atrophy whenadministered at an oral dose of greater than about 750 mg per day in ahuman. In a yet further aspect, the disclosed compounds inhibit muscleatrophy when administered at an oral dose of greater than about 1000 mgper day in a human. In a still further aspect, the disclosed compoundsinhibit muscle atrophy when administered at an oral dose of greater thanabout 1500 mg per day in a human. In an even further aspect, thedisclosed compounds inhibit muscle atrophy when administered at an oraldose of greater than about 2000 mg per day in a human.

It is contemplated that one or more compounds can optionally be omittedfrom the disclosed invention.

C. PHARMACEUTICAL COMPOSITIONS

In one aspect, the invention relates to pharmaceutical compositionscomprising the disclosed compounds. That is, a pharmaceuticalcomposition can be provided comprising a therapeutically effectiveamount of at least one disclosed compound. In another example, apharmaceutical composition can be provided comprising a prophylacticallyeffective amount of at least one disclosed compound

In one aspect, the invention relates to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a compound, whereinthe compound is present in an effective amount. The compound can beselected from a tacrine analog, allantoin analog, naringenin analog,conessine analog, tomatidine analog, ungerine/hippeastrine analog andbetulinic acid analog. For example, the compound can be a tacrineanalog. In another example, the compound can be a naringenin analog. Inanother example, the compound can be a conessine analog. In anotherexample, the compound can be a tomatidine analog. In another example,the compound can be an ungerine/hippeastrine analog. In another example,the compound can be a betulinic acid analog.

In one aspect, the compound is present in an amount greater than aboutan amount selected from 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200mg, 250 mg, 300 mg, 400, mg, 500 mg, 750 mg, 1000 mg, 1,500 mg, or 2,000mg.

A pharmaceutical composition comprising a pharmaceutically acceptablecarrier and an effective amount of one or more of: (a) a compoundselected from a tacrine analog, allantoin analog, naringenin analog,conessine analog, tomatidine analog, ungerine/hippeastrine analog andbetulinic acid analog; (b) a compound that down regulates multipleinduced mRNAs of Muscle Atrophy Signature 1, compared to expressionlevels in the same type of the muscle cell in the absence of thecompound; (c) a compound that up regulates multiple repressed mRNAs ofMuscle Atrophy Signature 1, compared to expression levels in the sametype of the muscle cell in the absence of the compound; (d) a compoundthat down regulates multiple induced mRNAs of Muscle Atrophy Signature2, compared to expression levels in the same type of the muscle cell inthe absence of the compound; and/or (e) a compound that up regulatesmultiple mRNAs of Muscle Atrophy Signature 2, compared to expressionlevels in the same type of the muscle cell in the absence of thecompound.

In a further aspect, the amount is a therapeutically effective amount.In a still further aspect, the amount is a prophylactically effectiveamount.

In a further aspect, pharmaceutical composition is administered to ananimal. In a still further aspect, the animal is a mammal, fish or bird.In a yet further aspect, the mammal is a primate. In a still furtheraspect, the mammal is a human. In an even further aspect, the human is apatient.

In a further aspect, the pharmaceutical composition comprises a compoundidentified using muscle atrophy signature-1. In a yet further aspect,the pharmaceutical composition comprises a compound identified usingmuscle atrophy signature-2. In a yet further aspect, the pharmaceuticalcomposition comprises a compound identified using both muscle atrophysignature-1 and muscle atrophy signature-2.

In a further aspect, the animal is a domesticated animal. In a stillfurther aspect, the domesticated animal is a domesticated fish,domesticated crustacean, or domesticated mollusk. In a yet furtheraspect, the domesticated animal is poultry. In an even further aspect,the poultry is selected from chicken, turkey, duck, and goose. In astill further aspect, the domesticated animal is livestock. In a yetfurther aspect, the livestock animal is selected from pig, cow, horse,goat, bison, and sheep.

In a further aspect, the effective amount is a therapeutically effectiveamount. In a still further aspect, the effective amount is aprophylactically effective amount. In a yet further aspect, the muscledisorder is muscle atrophy. In an even further aspect, the muscledisorder is a condition in need of promoting muscle health, promotingnormal muscle function, and/or promoting healthy aging muscles.

In a further aspect, the pharmaceutical composition is administeredfollowing identification of the mammal in need of treatment of muscleatrophy. In a still further aspect, the pharmaceutical composition isadministered following identification of the mammal in need ofprevention of muscle atrophy. In an even further aspect, the mammal hasbeen diagnosed with a need for treatment of muscle atrophy prior to theadministering step.

In certain aspects, the disclosed pharmaceutical compositions comprisethe disclosed compounds (including pharmaceutically acceptable salt(s)thereof) as an active ingredient, a pharmaceutically acceptable carrier,and, optionally, other therapeutic ingredients or adjuvants. The instantcompositions include those suitable for oral, rectal, topical, andparenteral (including subcutaneous, intramuscular, and intravenous)administration, although the most suitable route in any given case willdepend on the particular host, and nature and severity of the conditionsfor which the active ingredient is being administered. Thepharmaceutical compositions can be conveniently presented in unit dosageform and prepared by any of the methods well known in the art ofpharmacy.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts prepared from pharmaceutically acceptable non-toxic bases oracids. When the compound of the present invention is acidic, itscorresponding salt can be conveniently prepared from pharmaceuticallyacceptable non-toxic bases, including inorganic bases and organic bases.Salts derived from such inorganic bases include aluminum, ammonium,calcium, copper (-ic and -ous), ferric, ferrous, lithium, magnesium,manganese (-ic and -ous), potassium, sodium, zinc and the like salts.Particularly preferred are the ammonium, calcium, magnesium, potassiumand sodium salts. Salts derived from pharmaceutically acceptable organicnon-toxic bases include salts of primary, secondary, and tertiaryamines, as well as cyclic amines and substituted amines such asnaturally occurring and synthesized substituted amines. Otherpharmaceutically acceptable organic non-toxic bases from which salts canbe formed include ion exchange resins such as, for example, arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like.

As used herein, the term “pharmaceutically acceptable non-toxic acids”,includes inorganic acids, organic acids, and salts prepared thereof, forexample, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric,isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic,nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric,p-toluenesulfonic acid and the like. Preferred are citric, hydrobromic,hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

In practice, the compounds of the invention, or pharmaceuticallyacceptable salts thereof, of this invention can be combined as theactive ingredient in intimate admixture with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier can take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., oral or parenteral(including intravenous). Thus, the pharmaceutical compositions of thepresent invention can be presented as discrete units suitable for oraladministration such as capsules, cachets or tablets each containing apredetermined amount of the active ingredient. Further, the compositionscan be presented as a powder, as granules, as a solution, as asuspension in an aqueous liquid, as a non-aqueous liquid, as anoil-in-water emulsion or as a water-in-oil liquid emulsion. In additionto the common dosage forms set out above, the compounds of theinvention, and/or pharmaceutically acceptable salt(s) thereof, can alsobe administered by controlled release means and/or delivery devices. Thecompositions can be prepared by any of the methods of pharmacy. Ingeneral, such methods include a step of bringing into association theactive ingredient with the carrier that constitutes one or morenecessary ingredients. In general, the compositions are prepared byuniformly and intimately admixing the active ingredient with liquidcarriers or finely divided solid carriers or both. The product can thenbe conveniently shaped into the desired presentation.

Thus, the pharmaceutical compositions of this invention can include apharmaceutically acceptable carrier and a compound or a pharmaceuticallyacceptable salt of the compounds of the invention. The compounds of theinvention, or pharmaceutically acceptable salts thereof, can also beincluded in pharmaceutical compositions in combination with one or moreother therapeutically active compounds.

The pharmaceutical carrier employed can be, for example, a solid,liquid, or gas. Examples of solid carriers include lactose, terra alba,sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, andstearic acid. Examples of liquid carriers are sugar syrup, peanut oil,olive oil, and water. Examples of gaseous carriers include carbondioxide and nitrogen.

In preparing the compositions for oral dosage form, any convenientpharmaceutical media can be employed. For example, water, glycols, oils,alcohols, flavoring agents, preservatives, coloring agents and the likecan be used to form oral liquid preparations such as suspensions,elixirs and solutions; while carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents, and the like can be used to form oralsolid preparations such as powders, capsules and tablets. Because oftheir ease of administration, tablets and capsules are the preferredoral dosage units whereby solid pharmaceutical carriers are employed.Optionally, tablets can be coated by standard aqueous or nonaqueoustechniques

A tablet containing the composition of this invention can be prepared bycompression or molding, optionally with one or more accessoryingredients or adjuvants. Compressed tablets can be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets can be made by molding in a suitable machine, a mixtureof the powdered compound moistened with an inert liquid diluent.

The pharmaceutical compositions of the present invention comprise acompound of the invention (or pharmaceutically acceptable salts thereof)as an active ingredient, a pharmaceutically acceptable carrier, andoptionally one or more additional therapeutic agents or adjuvants. Theinstant compositions include compositions suitable for oral, rectal,topical, and parenteral (including subcutaneous, intramuscular, andintravenous) administration, although the most suitable route in anygiven case will depend on the particular host, and nature and severityof the conditions for which the active ingredient is being administered.The pharmaceutical compositions can be conveniently presented in unitdosage form and prepared by any of the methods well known in the art ofpharmacy.

Pharmaceutical compositions of the present invention suitable forparenteral administration can be prepared as solutions or suspensions ofthe active compounds in water. A suitable surfactant can be includedsuch as, for example, hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofin oils. Further, a preservative can be included to prevent thedetrimental growth of microorganisms.

Pharmaceutical compositions of the present invention suitable forinjectable use include sterile aqueous solutions or dispersions.Furthermore, the compositions can be in the form of sterile powders forthe extemporaneous preparation of such sterile injectable solutions ordispersions. In all cases, the final injectable form must be sterile andmust be effectively fluid for easy syringability. The pharmaceuticalcompositions must be stable under the conditions of manufacture andstorage; thus, preferably should be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol and liquid polyethyleneglycol), vegetable oils, and suitable mixtures thereof.

Pharmaceutical compositions of the present invention can be in a formsuitable for topical use such as, for example, an aerosol, cream,ointment, lotion, dusting powder, mouth washes, gargles, and the like.Further, the compositions can be in a form suitable for use intransdermal devices. These formulations can be prepared, utilizing acompound of the invention, or pharmaceutically acceptable salts thereof,via conventional processing methods. As an example, a cream or ointmentis prepared by mixing hydrophilic material and water, together withabout 5 wt % to about 10 wt % of the compound, to produce a cream orointment having a desired consistency.

Pharmaceutical compositions of this invention can be in a form suitablefor rectal administration wherein the carrier is a solid. It ispreferable that the mixture forms unit dose suppositories. Suitablecarriers include cocoa butter and other materials commonly used in theart. The suppositories can be conveniently formed by first admixing thecomposition with the softened or melted carrier(s) followed by chillingand shaping in moulds.

In addition to the aforementioned carrier ingredients, thepharmaceutical formulations described above can include, as appropriate,one or more additional carrier ingredients such as diluents, buffers,flavoring agents, binders, surface-active agents, thickeners,lubricants, preservatives (including anti-oxidants) and the like.Furthermore, other adjuvants can be included to render the formulationisotonic with the blood of the intended recipient. Compositionscontaining a compound of the invention, and/or pharmaceuticallyacceptable salts thereof, can also be prepared in powder or liquidconcentrate form.

In the treatment conditions which require modulation of cellularfunction related to muscle health, muscle function and/or healthy muscleaging an appropriate dosage level will generally be about 0.01 to 500 mgper kg patient body weight per day and can be administered in single ormultiple doses. Preferably, the dosage level will be about 0.1 to about250 mg/kg per day; more preferably 0.5 to 100 mg/kg per day. A suitabledosage level can be about 0.01 to 250 mg/kg per day, about 0.05 to 100mg/kg per day, or about 0.1 to 50 mg/kg per day. Within this range thedosage can be 0.05 to 0.5, 0.5 to 5.0 or 5.0 to 50 mg/kg per day. Fororal administration, the compositions are preferably provided in thefrom of tablets containing 1.0 to 1000 milligrams of the activeingredient, particularly 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150,200, 250, 300, 400, 500, 600, 750, 800, 900 and 1000 milligrams of theactive ingredient for the symptomatic adjustment of the dosage of thepatient to be treated. The compound can be administered on a regimen of1 to 4 times per day, preferably once or twice per day. This dosingregimen can be adjusted to provide the optimal therapeutic response.

It is understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors. Such factorsinclude the age, body weight, general health, sex, and diet of thepatient. Other factors include the time and route of administration,rate of excretion, drug combination, and the type and severity of theparticular disease undergoing therapy.

The present invention is further directed to a method for themanufacture of a medicament for modulating cellular activity related tomuscle health, muscle function, and/or healthy aging muscles (e.g.,treatment of one or more disorders associated with muscle dysfunction oratrophy) in mammals (e.g., humans) comprising combining one or moredisclosed compounds, products, or compositions with a pharmaceuticallyacceptable carrier or diluent. Thus, in one aspect, the inventionrelates to a method for manufacturing a medicament comprising combiningat least one disclosed compound or at least one disclosed product with apharmaceutically acceptable carrier or diluent.

The disclosed pharmaceutical compositions can further comprise othertherapeutically active compounds, which are usually applied in thetreatment of the above mentioned pathological conditions.

It is understood that the disclosed compositions can be prepared fromthe disclosed compounds. It is also understood that the disclosedcompositions can be employed in the disclosed methods of using.

D. METHODS OF USING THE COMPOUNDS AND COMPOSITIONS

1. Muscle Atrophy

Muscle atrophy is defined as a decrease in the mass of the muscle; itcan be a partial or complete wasting away of muscle. When a muscleatrophies, this leads to muscle weakness, since the ability to exertforce is related to mass. Muscle atrophy is a co-morbidity of severalcommon diseases, and patients who have “cachexia” in these diseasesettings have a poor prognosis.

Muscle atrophy can also be skeletal muscle loss or weakness caused bymalnutrition, aging, muscle disuse (such as voluntary and involuntarybed rest, neurologic disease (such as multiple sclerosis, amyotrophiclateral sclerosis, spinal muscular atrophy, critical illness neuropathy,spinal cord injury, peripheral neuropathy, or peripheral nerve injury),injury to the limbs or joints, casting, other post-surgical forms oflimb immobilization, or spaceflight), chronic disease (such as cancer,congestive heart failure, chronic pulmonary disease, chronic renalfailure, chronic liver disease, diabetes mellitus, glucocorticoidexcess, growth hormone deficiency, IGF-I deficiency, estrogendeficiency, and chronic infections such as HIV/AIDS or tuberculosis),burn injuries, sepsis, other illnesses requiring mechanicalventiliation, drug-induced muscle disease (such asglucocorticoid-induced myopathy and statin-induced myopathy), geneticdiseases that primarily affect skeletal muscle (such as musculardystrophy, myotonic dystrophy and inclusion body myositis), orautoimmune diseases that affect skeletal muscle (such as polymyositisand dermatomyositis).

There are many diseases and conditions which cause muscle atrophy,including malnutrition, muscle disuse (secondary to voluntary orinvoluntary bed rest, neurologic disease (including multiple sclerosis,amyotrophic lateral sclerosis, spinal muscular atrophy, critical illnessneuropathy, spinal cord injury or peripheral nerve injury), orthopedicinjury, casting, and other post-surgical forms of limb immobilization),chronic disease (including cancer, congestive heart failure, chronicpulmonary disease, chronic renal failure, chronic liver disease,diabetes mellitus, Cushing syndrome, growth hormone deficiency, IGF-Ideficiency, estrogen deficiency, and chronic infections such as HIV/AIDSor tuberculosis), burns, sepsis, other illnesses requiring mechanicalventilation, drug-induced muscle disease (such as glucorticoid-inducedmyopathy and statin-induced myopathy), genetic diseases that primarilyaffect skeletal muscle (such as muscular dystrophy and myotonicdystrophy), autoimmune diseases that affect skeletal muscle (such aspolymyositis and dermatomyositis), spaceflight, and aging.

Muscle atrophy occurs by a change in the normal balance between proteinsynthesis and protein degradation. During atrophy, there is adown-regulation of protein synthesis pathways, and an activation ofprotein breakdown pathways. The particular protein degradation pathwaywhich seems to be responsible for much of the muscle loss seen in amuscle undergoing atrophy is the ATP-dependent, ubiquitin/proteasomepathway. In this system, particular proteins are targeted fordestruction by the ligation of at least four copies of a small peptidecalled ubiquitin onto a substrate protein. When a substrate is thus“poly-ubiquitinated,” it is targeted for destruction by the proteasome.Particular enzymes in the ubiquitin/proteasome pathway allowubiquitination to be directed to some proteins but notothers—specificity is gained by coupling targeted proteins to an “E3ubiquitin ligase.” Each E3 ubiquitin ligase binds to a particular set ofsubstrates, causing their ubiquitination. For example, in skeletalmuscle, the E3 ubiquitin ligases atrogin-1 and MuRF1 are known to playessential roles protein degradation and muscle atrophy.

Muscle atrophy can be opposed by the signaling pathways which inducemuscle hypertrophy, or an increase in muscle size. Therefore one way inwhich exercise induces an promote muscle health, promote normal musclefunction, and/or promote healthy aging muscles is to downregulate thepathways which have the opposite effect. One important rehabilitationtool for muscle atrophy includes the use of functional electricalstimulation to stimulate the muscles which has had limited success inthe rehabilitation of paraplegic patients.

In certain aspects, the disclosed compounds can be used as a therapy forillness- and age-related muscle atrophy. It can be useful as amonotherapy or in combination with other strategies that have beenconsidered, such as myostatin inhibition (Zhou, X., et al. (2010) Cell142(4): 531-543). Given its capacity to reduce adiposity, fasting bloodglucose and plasma lipid levels, a disclosed compound derivatives canalso be used as a therapy for obesity, metabolic syndrome and type 2diabetes.

The disclosed compounds can be used as single agents or in combinationwith one or more other drugs in the treatment, prevention, control,amelioration or reduction of risk of the aforementioned diseases,disorders and conditions for which compounds of formula I or the otherdrugs have utility, where the combination of drugs together are safer ormore effective than either drug alone. The other drug(s) can beadministered by a route and in an amount commonly used therefore,contemporaneously or sequentially with a disclosed compound. When adisclosed compound is used contemporaneously with one or more otherdrugs, a pharmaceutical composition in unit dosage form containing suchdrugs and the disclosed compound is preferred. However, the combinationtherapy can also be administered on overlapping schedules. It is alsoenvisioned that the combination of one or more active ingredients and adisclosed compound will be more efficacious than either as a singleagent.

Systemic administration of one or more disclosed compounds (e.g., byparenteral injection or by oral consumption) can be used to promotemuscle health, promote normal muscle function, and/or promote healthyaging muscles, and reduce muscle atrophy in all muscles, including thoseof the limbs and the diaphragm. Local administration of a disclosedcompound (by a topical route or localized injection) can be used topromote local muscle health, as can be required following a localizedinjury or surgery.

In one aspect, the subject compounds can be coadministered with agentsthat stimulate insulin signaling, IGF1 signaling and/or muscle healthincluding ursolic acid, insulin, insulin analogs, insulin-like growthfactor 1, metformin, thiazoladinediones, sulfonylureas, meglitinides,leptin, dipeptidyl peptidase-4 inhibitors, glucagon-like peptide-Iagonists, tyrosine-protein phosphatase non-receptor type inhibitors,myostatin signaling inhibitors, beta-2 adrenergic agents includingclenbuterol, androgens, selective androgen receptor modulator (such asGTx-024, BMS-564,929, LGD-4033, AC-262,356, JNJ-28330835, LGD-2226,LGD-3303, S-40503, or S-23), aromatase inhibitors (such as anastrozole,letrozole, exemestane, vorozole, formestane, fadrozole,4-hydroxyandrostenedione, 1,4,6-androstatrien-3,17-dione, and4-androstene-3,6,17-trione), growth hormone, a growth hormone analog,ghrelin, a ghrelin analog. A disclosed compound or salt thereof can beadministered orally, intramuscularly, intravenously or intraarterially.A disclosed compound or salt thereof can be substantially pure. Adisclosed compound or salt thereof can be administered at about 10mg/day to 10 g/day.

In another aspect, the subject compounds can be administered incombination with agents that stimulate ursolic acid, insulin, insulinanalogs, insulin-like growth factor 1, metformin, thiazoladinediones,sulfonylureas, meglitinides, leptin, dipeptidyl peptidase-4 inhibitors,glucagon-like peptide-1 agonists, tyrosine-protein phosphatasenon-receptor type inhibitors, myostatin signaling inhibitors, beta-2adrenergic agents including clenbuterol, androgens, selective androgenreceptor modulator (such as GTx-024, BMS-564,929, LGD-4033, AC-262,356,JNJ-28330835, LGD-2226, LGD-3303, S-40503, or S-23), aromataseinhibitors (such as anastrozole, letrozole, exemestane, vorozole,formestane, fadrozole, 4-hydroxyandrostenedione,1,4,6-androstatrien-3,17-dione, and 4-androstene-3,6,17-trione), growthhormone, a growth hormone analog, ghrelin, or a ghrelin analog. Adisclosed compound or salt thereof can be administered orally,intramuscularly, intravenously or intraarterially. A disclosed compoundor salt thereof can be substantially pure. A disclosed compound or saltthereof can be administered at about 10 mg/day to 10 g/day.

The pharmaceutical compositions and methods of the present invention canfurther comprise other therapeutically active compounds as noted hereinwhich are usually applied in the treatment of the above mentionedpathological conditions.

2. Treatment Methods

The compounds disclosed herein are useful for treating, preventing,ameliorating, controlling or reducing the risk of a variety of muscledisorders. Examples of such muscle disorders include, but are notlimited to, skeletal muscle atrophy secondary to malnutrition, muscledisuse (secondary to voluntary or involuntary bedrest), neurologicdisease (including multiple sclerosis, amyotrophic lateral sclerosis,spinal muscular atrophy, critical illness neuropathy, spinal cord injuryor peripheral nerve injury), orthopedic injury, casting, and otherpost-surgical forms of limb immobilization, chronic disease (includingcancer, congestive heart failure, chronic pulmonary disease, chronicrenal failure, chronic liver disease, diabetes mellitus, Cushingsyndrome and chronic infections such as HIV/AIDS or tuberculosis),burns, sepsis, other illnesses requiring mechanical ventiliation,drug-induced muscle disease (such as glucorticoid-induced myopathy andstatin-induced myopathy), genetic diseases that primarily affectskeletal muscle (such as muscular dystrophy and myotonic dystrophy),autoimmune diseases that affect skeletal muscle (such as polymyositisand dermatomyositis), spaceflight, or age-related sarcopenia. In stillfurther aspects, the invention is related to methods to modulate musclehealth, methods to inhibit muscle atrophy.

Thus, provided is a method for treating or preventing muscle atrophy,comprising: administering to a subject at least one disclosed compound;at least one disclosed pharmaceutical composition; and/or at least onedisclosed product in a dosage and amount effective to treat the disorderin the subject.

Also provided is a method for promoting muscle health, promote normalmuscle function, and/or promote healthy aging muscles comprising:administering to a subject at least one disclosed compound; at least onedisclosed pharmaceutical composition; and/or at least one disclosedproduct in a dosage and amount effective to treat the disorder in thesubject.

The compounds disclosed herein are useful for treating, preventing,ameliorating, controlling or reducing the risk of a variety of metabolicdisorders. In a further aspect, the disclosed compounds in treatingdisorders associated with a dysfunction of insulin/IGF-I signaling.Thus, are provided methods to increase insulin/IGF-I signaling, methodsto reduce body fat; methods to reduce blood glucose, methods to reduceblood triglycerides, methods to reduce blood cholesterol, methods toreduce obesity, methods to reduce fatty liver disease, and methods toreduce diabetes, and pharmaceutical compositions comprising compoundsused in the methods.

a. Treating Muscle Atrophy

Disclosed herein is a method of treating muscle atrophy in an animalcomprising administering to the animal an effective amount of acompound. The compound can be selected from a tacrine and analogs,naringenin and analogs, allantoin and analogs, conessine and analogs,tomatidine and analogs, ungerine/hippeastrine and analogs, and betulinicacid and analogs, or a mixture thereof. For example, the compound can bea tacrine analog. In another example, the compound can be a naringeninanalog. In another example, the compound can be an allantoin analog. Inanother example, the compound can be a conessine analog. In anotherexample, the compound can be a tomatidine analog. In another example,the compound can be a ungerine/hippeastrine analog. In another example,the compound can be a betulinic acid analog.

In one aspect, the compound is administered in an amount between about0.01 to 500 mg per kg patient body weight per day and can beadministered in single or multiple doses. Preferably, the dosage levelwill be about 0.1 to about 250 mg/kg per day; more preferably 0.5 to 100mg/kg per day. A suitable dosage level can be about 0.01 to 250 mg/kgper day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg perday. Within this range the dosage can be 0.05 to 0.5, 0.5 to 5.0 or 5.0to 50 mg/kg per day. For oral administration, the compositions arepreferably provided in the from of tablets containing 1.0 to 1000milligrams of the active ingredient, particularly 1.0, 5.0, 10, 15, 20,25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900 and1000 milligrams of the active ingredient for the symptomatic adjustmentof the dosage of the patient to be treated. The compound can beadministered on a regimen of 1 to 4 times per day, preferably once ortwice per day. This dosing regimen can be adjusted to provide theoptimal therapeutic response

In one aspect, the disclosed compounds inhibit muscle atrophy. In afurther aspect, the disclosed compounds promote muscle health, promotenormal muscle function, and/or promote healthy aging muscles. In a yetfurther aspect, the disclosed compounds inhibit of muscle atrophy andpromoting muscle health, promoting normal muscle function, and/orpromoting healthy aging muscles. In an even further aspect, thedisclosed compounds inhibit of muscle atrophy.

In a further aspect, the compound administered is a disclosed compoundor a product of a disclosed method of making a compound. In a yetfurther aspect, the invention relates to a pharmaceutical compositioncomprising at least one compound as disclosed herein.

In a further aspect, the compound is co-administered with an anabolicagent. In a further aspect, wherein the compound is co-administered withursolic acid or a ursolic acid derivative.

In a further aspect, the animal is a mammal, fish or bird. In a yetfurther aspect, the mammal is a primate. In a still further aspect, themammal is a human. In an even further aspect, the human is a patient.

In a further aspect, the Muscle Atrophy Signature is Muscle AtrophySignature 1. In a still further aspect, the Muscle Atrophy Signature isMuscle Atrophy Signature 2.

In a further aspect, prior to the administering step the mammal has beendiagnosed with a need for treatment of a disorder selected muscleatrophy, diabetes, obesity, and fatty liver disease. In a yet furtheraspect, the disorder is muscle atrophy.

In a further aspect, prior to the administering step the mammal has beendiagnosed with a need for treatment of a disorder associated with adysfunction in insulin/IGF-I signaling.

In a further aspect, the treatment of the disorder increases muscleIGF-I signaling. In a still further aspect, the treatment of thedisorder increases muscle IGF-I production.

In a further aspect, prior to the administering step the mammal has beendiagnosed with a need for treatment of a disorder associated withcirculating levels of leptin. In a still further aspect, the treatmentdecreases the circulating levels of leptin.

In a further aspect, administration the methods are promoting musclehealth, promoting normal muscle function, and/or promoting healthy agingmuscles in the mammal. In a yet further aspect, administration increasesenergy expenditure. In a still further aspect, increases brown fat. Inan even further aspect, administration increases the ratio of brown fatto white fat. In a still further aspect, administration increases theratio of skeletal muscle to fat. In a yet further aspect, the compoundis co-administered with a disclosed compound or a derivative thereof.

In a further aspect, the animal is a domesticated animal. In a stillfurther aspect, the domesticated animal is a domesticated fish,domesticated crustacean, or domesticated mollusk. In a yet furtheraspect, the domesticated animal is poultry. In an even further aspect,the poultry is selected from chicken, turkey, duck, and goose. In astill further aspect, the domesticated animal is livestock. In a yetfurther aspect, the livestock animal is selected from pig, cow, horse,goat, bison, and sheep.

In a further aspect, the effective amount is a therapeutically effectiveamount. In a still further aspect, the effective amount is aprophylactically effective amount. In a yet further aspect, muscleatrophy is prevented by administration of the compound. In an evenfurther aspect, muscle atrophy is treated by administration of thecompound. In a still further aspect, the method further comprises thestep of identifying the mammal in need of treatment of muscle atrophy.In a yet further aspect, the method further comprises the step ofidentifying the mammal in a need of prevention of muscle atrophy. In aneven further aspect, the mammal has been diagnosed with a need fortreatment of muscle atrophy prior to the administering step.

b. Promoting Muscle Health

In one aspect, the invention relates to a method for promoting musclehealth, promoting normal muscle function, and/or promoting healthy agingmuscles in an animal, the method comprising administering to the animalan effective amount of a compound selected from a tacrine and analogs,naringenin and analogs, allantoin and analogs, conessine and analogs,tomatidine and analogs, ungerine/hippeastrine and analogs, and betulinicacid and analogs, or a mixture thereof, thereby promoting muscle healthin the animal. For example, the compound can be a tacrine analog. Inanother example, the compound can be a naringenin analog. In anotherexample, the compound can be an allantoin analog. In another example,the compound can be a conessine analog. In another example, the compoundcan be a tomatidine analog. In another example, the compound can be aungerine/hippeastrine analog. In another example, the compound can be abetulinic acid analog. In one aspect, the invention relates to a methodfor promoting muscle health. In another aspect, the invention relates toa method for promoting normal muscle function. In another aspect, theinvention relates to a method for promoting healthy aging muscles.

In one aspect, the invention relates to a method for promoting musclehealth, promoting normal muscle function, and/or promoting healthy agingmuscles in an animal, the method comprising administering to the animalan effective amount of a compound, wherein the compound down regulatesat least one of the induced mRNAs of Muscle Atrophy Signature 1 orMuscle Atrophy Signature 2, compared to expression levels in the sametype of the muscle cell in the absence of the compound, and/or whereinthe compound up regulates at least one of the repressed mRNAs of MuscleAtrophy Signature 1 or Muscle Atrophy Signature 2, compared toexpression levels in the same type of the muscle cell in the absence ofthe compound, thereby promoting muscle health, promoting normal musclefunction, and/or promoting healthy aging muscles in the animal.

In a further aspect, the animal is a mammal, fish or bird. In a yetfurther aspect, the mammal is a primate. In a still further aspect, themammal is a human. In an even further aspect, the human is a patient.

In a further aspect, the Muscle Atrophy Signature is Muscle AtrophySignature 1. In a still further aspect, the Muscle Atrophy Signature isMuscle Atrophy Signature 2.

In a further aspect, prior to the administering step the mammal has beendiagnosed with a need for treatment of a disorder selected muscleatrophy, diabetes, obesity, and fatty liver disease. In a yet furtheraspect, the disorder is muscle atrophy.

In a further aspect, prior to the administering step the mammal has beendiagnosed with a need for treatment of a disorder associated with adysfunction in insulin/IGF-I signaling.

In a further aspect, the treatment of the disorder increases muscleIGF-I signaling. In a still further aspect, the treatment of thedisorder increases muscle IGF-I production.

In a further aspect, prior to the administering step the mammal has beendiagnosed with a need for treatment of a disorder associated withcirculating levels of leptin. In a still further aspect, the treatmentdecreases the circulating levels of leptin.

In a further aspect, administration promoting muscle health, promotingnormal muscle function, and/or promoting healthy aging muscles in themammal. In a yet further aspect, administration increases energyexpenditure. In a still further aspect, increases brown fat. In an evenfurther aspect, administration increases the ratio of brown fat to whitefat. In a still further aspect, administration increases the ratio ofskeletal muscle to fat. In a yet further aspect, the compound isco-administered with a disclosed compound or a derivative thereof.

In a further aspect. the animal is a domesticated animal. In a stillfurther aspect, the domesticated animal is a domesticated fish,domesticated crustacean, or domesticated mollusk. In a yet furtheraspect, the domesticated animal is poultry. In an even further aspect,the poultry is selected from chicken, turkey, duck, and goose. In astill further aspect, the domesticated animal is livestock. In a yetfurther aspect, the livestock animal is selected from pig, cow, horse,goat, bison, and sheep.

In a further aspect, the effective amount is a therapeutically effectiveamount. In a still further aspect, the effective amount is aprophylactically effective amount. In a yet further aspect, muscleatrophy is prevented by administration of the compound. In an evenfurther aspect, muscle atrophy is treated by administration of thecompound. In a still further aspect, the method further comprises thestep of identifying the mammal in need of treatment of muscle atrophy.In a yet further aspect, the method further comprises the step ofidentifying the mammal in a need of prevention of muscle atrophy. In aneven further aspect, the mammal has been diagnosed with a need fortreatment of muscle atrophy prior to the administering step.

c. Enhancing Muscle Formation

In one aspect, the invention relates to a method of enhancing muscleformation in a mammal, the method comprising administering to the animalan effective amount of a compound selected from a tacrine and analogs,naringenin and analogs, allantoin and analogs, conessine and analogs,tomatidine and analogs, ungerine/hippeastrine and analogs, and betulinicacid and analogs, or a mixture thereof, thereby promoting muscle health,promoting normal muscle function, and/or promoting healthy aging musclesin the animal. For example, the compound can be a tacrine analog. Inanother example, the compound can be a naringenin analog. In anotherexample, the compound can be an allantoin analog. In another example,the compound can be a conessine analog. In another example, the compoundcan be a tomatidine analog. In another example, the compound can be aungerine/hippeastrine analog. In another example, the compound can be abetulinic acid analog.

In a further aspect, the invention relates to a method of enhancingmuscle formation in a mammal, the method comprising administering to theanimal an effective amount of a compound, wherein the compound downregulates at least one of the induced mRNAs of Muscle Atrophy Signature1 or Muscle Atrophy Signature 2, compared to expression levels in thesame type of the muscle cell in the absence of the compound, and/orwherein the compound up regulates at least one of the repressed mRNAs ofMuscle Atrophy Signature 1 or Muscle Atrophy Signature 2, compared toexpression levels in the same type of the muscle cell in the absence ofthe compound, thereby promoting muscle health, promoting normal musclefunction, and/or promoting healthy aging muscles in the animal.

In a further aspect, the mammal is a human. In a still further aspect,the human is a patient. In a yet further aspect, administration of thecompound prevents muscle atrophy in the mammal. In an even furtheraspect, administration of the compound treats muscle atrophy in themammal. In a still further aspect, administration of the compoundpromote muscle health, promote normal muscle function, and/or promotehealthy aging muscles in the mammal.

In a further aspect, the compound is administered in an effectiveamount. In a yet further aspect, the effective amount is atherapeutically effective amount. In a still further aspect, theeffective amount is a prophylactically effective amount. In a stillfurther aspect, the method further comprises the step of identifying themammal in need of treatment of muscle atrophy. In a yet further aspect,the method further comprises the step of identifying the mammal in needof prevention of muscle atrophy. In an even further aspect, the mammalhas been diagnosed with a need for treatment of muscle atrophy prior tothe administering step.

In a further aspect. the mammal is a domesticated animal. In a yetfurther aspect, domesticated animal is livestock. In a yet furtheraspect, the livestock animal is selected from pig, cow, horse, goat,bison, and sheep.

3. Facilitating Tissue Formation In Vitro

In one aspect, the invention relates to a method of enhancing tissuehealth in vitro, the method comprising administering to the tissue aneffective amount of a compound wherein the compound down regulates atleast one of the induced mRNAs of Muscle Atrophy Signature 1 or MuscleAtrophy Signature 2, compared to expression levels in the same type ofthe muscle cell in the absence of the compound, and/or wherein thecompound up regulates at least one of the repressed mRNAs of MuscleAtrophy Signature 1 or Muscle Atrophy Signature 2, compared toexpression levels in the same type of the muscle cell in the absence ofthe compound, thereby promoting muscle health, promoting normal musclefunction, and/or promoting healthy aging muscles.

In a further aspect, the compound administered is a disclosed compound.In a further aspect, the compound is selected from a tacrine andanalogs, naringenin and analogs, allantoin and analogs, conessine andanalogs, tomatidine and analogs, ungerine/hippeastrine and analogs, andbetulinic acid and analogs, or a mixture thereof, thereby facilitatingtissue formation in vitro. For example, the compound can be a tacrineanalog. In another example, the compound can be a naringenin analog. Inanother example, the compound can be an allantoin analog. In anotherexample, the compound can be a conessine analog. In another example, thecompound can be a tomatidine analog. In another example, the compoundcan be a ungerine/hippeastrine analog. In another example, the compoundcan be a betulinic acid analog.

In a further aspect, the tissue comprises animal cells. In a stillfurther aspect, the animal cells are muscle cells. In a yet furtheraspect, the muscle cells are skeletal muscle stem or progenitor cells.In an even further aspect, the skeletal muscle stem or progenitor cellsare grown on a scaffold.

4. Manufacture of a Medicament

In one aspect, the invention relates to a method for the manufacture ofa medicament for inhibiting muscle atrophy and for promoting musclehealth, promoting normal muscle function, and/or promoting healthy agingmuscles in a mammal comprising combining a therapeutically effectiveamount of a disclosed compound or product of a disclosed method with apharmaceutically acceptable carrier or diluent.

In one aspect, the invention relates to a method for manufacturing amedicament associated with muscle atrophy or the need to promote musclehealth, promote normal muscle function, and/or promote healthy agingmuscles, the method comprising the step of combining an effective amountof one or more of: (a) a compound selected from tacrine analog,naringenin analog, allantoin analog, conessine analog, tomatidineanalog, ungerine/hippeastrine analog and betulinic acid analog, or amixture thereof; (b) a compound that down regulates multiple inducedmRNAs of Muscle Atrophy Signature 1, compared to expression levels inthe same type of the muscle cell in the absence of the compound; (c) acompound that up multiple repressed mRNAs of Muscle Atrophy Signature 1,compared to expression levels in the same type of the muscle cell in theabsence of the compound; (d) a compound that down regulates multipleinduced mRNAs of Muscle Atrophy Signature 2, compared to expressionlevels in the same type of the muscle cell in the absence of thecompound; and/or (e) a compound that up regulates at least one of therepressed mRNAs of Muscle Atrophy Signature 2, compared to expressionlevels in the same type of the muscle cell in the absence of thecompound, with a pharmaceutically acceptable carrier or diluent.

In a further aspect, the medicament comprises a disclosed compound. In astill further aspect, the compound is selected from a tacrine andanalogs, naringenin and analogs, allantoin and analogs, conessine andanalogs, tomatidine and analogs, ungerine/hippeastrine and analogs, andbetulinic acid and analogs, or a mixture thereof. For example, thecompound can be a tacrine analog. In another example, the compound canbe a naringenin analog. In another example, the compound can be anallantoin analog. In another example, the compound can be a conessineanalog. In another example, the compound can be a tomatidine analog. Inanother example, the compound can be a ungerine/hippeastrine analog. Inanother example, the compound can be a betulinic acid analog.

In a further aspect, the medicament is modulates muscle health. In astill further aspect, the medicament inhibits muscle atrophy. In a yetfurther aspect, the medicament promote muscle health, promote normalmuscle function, and/or promote healthy aging muscles.

5. Kits

Also disclosed herein are kit comprising a tacrine analog, naringeninanalog, allantoin analog, conessine analog, tomatidine analog,ungerine/hippeastrine analog and betulinic acid analog, or a mixturethereof, and one or more of: a) at least one agent known to treat muscleatrophy in an animal; b) at least one agent known to decrease the riskof obtaining muscle atrophy in an animal; c) at least one agent known tohave a side effect of muscle atrophy; d) instructions for treatingmuscle atrophy; or e) at least one anabolic agent. For example, thecompound can be a tacrine analog. In another example, the compound canbe a naringenin analog. In another example, the compound can be aallantoin analog. In another example, the compound can be a conessineanalog. In another example, the compound can be a tomatidine analog. Inanother example, the compound can be a ungerine/hippeastrine analog. Inanother example, the compound can be a betulinic acid analog.

In one aspect, the kit further comprises at least one agent, wherein thecompound and the agent are co-formulated.

In another aspect, the compound and the agent are co-packaged. The agentcan be any agent as disclosed herein, such as anabolic agent, agentknown to have a side effect of muscle atrophy, agent known to decreasethe risk of obtaining muscle atrophy in an animal, or agent known totreat muscle atrophy in an animal.

In one aspect, the invention relates to a kit comprising an effectiveamount of one or more of: (a) a compound selected from a tacrine analog,naringenin analog, allantoin analog, conessine analog, tomatidineanalog, ungerine/hippeastrine analog and betulinic acid analog; (b) acompound that down regulates multiple induced mRNAs of Muscle AtrophySignature 1, compared to expression levels in the same type of themuscle cell in the absence of the compound; (c) a compound that upregulates multiple repressed mRNAs of Muscle Atrophy Signature 1,compared to expression levels in the same type of the muscle cell in theabsence of the compound; (d) a compound that down regulates multipleinduced mRNAs of Muscle Atrophy Signature 2, compared to expressionlevels in the same type of the muscle cell in the absence of thecompound; and/or (e) a compound that up regulates multiple repressedmRNAs of Muscle Atrophy Signature 2, compared to expression levels inthe same type of the muscle cell in the absence of the compound, (f) andone or more of: (i) a protein supplement; (ii) an anabolic agent; (iii)a catabolic agent; (iv) a dietary supplement; (v) at least one agentknown to treat a disorder associated with muscle wasting; (vi)instructions for treating a disorder associated with cholinergicactivity; or (vii) instructions for using the compound to promote musclehealth, promote normal muscle function, and/or promote healthy agingmuscles.

The kits can also comprise compounds and/or products co-packaged,co-formulated, and/or co-delivered with other components. For example, adrug manufacturer, a drug reseller, a physician, a compounding shop, ora pharmacist can provide a kit comprising a disclosed compound and/orproduct and another component for delivery to a patient.

It is contemplated that the disclosed kits can be used in connectionwith the disclosed methods of making, the disclosed methods of using,and/or the disclosed compositions.

6. Method of Lowering Blood Glucose

In one aspect, the invention relates to a method of lowering bloodglucose in an animal comprising administering to the animal an effectiveamount of a composition comprising ursolic acid and a naringenin analog,thereby lowering the blood glucose in the animal. In one aspect, thenaringenin analog can be naringenin. In one aspect, the ursolic acid canbe a ursolic acid derivative.

In another aspect, invention relates to a method of lowering bloodglucose in an animal comprising administering to the animal an effectiveamount of a hippeastrine analog, thereby lowering the blood glucose inthe animal. In one aspect, the hippeastrine analog can be hippeastrine.

In another aspect, invention relates to a method of lowering bloodglucose in an animal comprising administering to the animal an effectiveamount of a conessine analog, thereby lowering the blood glucose in theanimal. In one aspect, the conessine analog can be conessine.

In a further aspect, the animal is a mammal, fish or bird. In a yetfurther aspect, the mammal is a primate. In a still further aspect, themammal is a human. In an even further aspect, the human is a patient.

In a further aspect, prior to the administering step the mammal has beendiagnosed with a need for treatment of a disorder associated with theneed of lowering blood glucose.

In a further aspect, prior to the administering step the mammal has beendiagnosed with a need for treatment of a disorder associated with adysfunction in insulin/IGF-I signaling.

In a further aspect, the treatment of the disorder increases muscleIGF-I signaling. In a still further aspect, the treatment of thedisorder increases muscle IGF-I production.

In a further aspect, prior to the administering step the mammal has beendiagnosed with a need for treatment of a disorder associated withcirculating levels of leptin. In a still further aspect, the treatmentdecreases the circulating levels of leptin.

In a further aspect. the animal is a domesticated animal. In a stillfurther aspect, the domesticated animal is a domesticated fish,domesticated crustacean, or domesticated mollusk. In a yet furtheraspect, the domesticated animal is poultry. In an even further aspect,the poultry is selected from chicken, turkey, duck, and goose. In astill further aspect, the domesticated animal is livestock. In a yetfurther aspect, the livestock animal is selected from pig, cow, horse,goat, bison, and sheep.

In a further aspect, the effective amount is a therapeutically effectiveamount. In a still further aspect, the effective amount is aprophylactically effective amount. In a yet further aspect, high bloodglucose is prevented by administration of the compound. In a stillfurther aspect, the method further comprises the step of identifying themammal in need of treatment of lowering of blood glucose. In a yetfurther aspect, the method further comprises the step of identifying themammal in a need of prevention the need of lowering blood glucose. In aneven further aspect, the mammal has been diagnosed with a need forlowering of blood glucose prior to the administering step.

7. Identification of Compounds that Inhibit Muscle Atrophy

Also disclosed are methods for identifying a compound that inhibitsmuscle atrophy when administered in a effective amount to a animal inneed of treatment thereof, the method comprising the steps of: (i)selecting a candidate compound; (ii) determining the effect of thecandidate compound on a cell's expression levels of a plurality ofinduced mRNAs and/or repressed mRNAs of a Muscle Atrophy Signature,wherein the candidate compound is identified as suitable for muscleatrophy inhibition if: (a) more than one of the induced mRNAs of theMuscle Atrophy Signature are down regulated, compared to expressionlevels of the induced mRNAs of the Muscle Atrophy Signature in the sametype of cell in the absence of the candidate compound; and/or (b) morethan one of the repressed mRNAs of the Muscle Atrophy Signature are upregulated, compared to expression levels of the repressed mRNAs of theMuscle Atrophy Signature in the same type of cell in the absence of thecandidate compound. In one aspect, the method further comprisesadministering the candidate compound to an animal. In yet anotheraspect, the method further comprises writing a report. In yet anotheraspect, the method further comprises reporting the results. In yetanother aspect, the method further comprises performing further tests onthe candidate compound, such as confirmatory tests. In yet anotheraspect, the method further comprises performing toxicity studies on thecandidate compound.

In a further aspect, the candidate compound comprises a disclosedcompound. In a still further aspect, the compound is selected from atacrine analog, naringenin analog, allantoin analog, conessine analog,tomatidine analog, ungerine/hippeastrine analog and betulinic acidanalog, as defined elsewhere herein. For example, the compound can be atacrine analog. In another example, the compound can be a naringeninanalog. In another example, the compound can be an allantoin analog. Inanother example, the compound can be a conessine analog. In anotherexample, the compound can be a tomatidine analog. In another example,the compound can be a ungerine/hippeastrine analog. In another example,the compound can be a betulinic acid analog.

In a further aspect, the animal is a mammal, fish or bird. In a yetfurther aspect, the mammal is a primate. In a still further aspect, themammal is a human. In an even further aspect, the human is a patient.

In a further aspect, the Muscle Atrophy Signature is Muscle AtrophySignature 1. In a still further aspect, the Muscle Atrophy Signature isMuscle Atrophy Signature 2.

In a further aspect, the Muscle Atrophy Signature is determinedaccording to steps comprising: a) determining mRNA expression levels ina muscle cell undergoing muscle atrophy, b) determining mRNA expressionlevels in a muscle cell not undergoing muscle atrophy, wherein an mRNAis determined to be part of the Muscle Atrophy Signature if: (a0 themRNA is up regulated in the muscle cell undergoing muscle atrophycompared to the muscle cell not undergoing muscle atrophy, or (b) themRNA is down regulated in the muscle cell undergoing muscle atrophycompared to the muscle cell not undergoing muscle atrophy.

In one aspect, the muscle cell undergoing atrophy and the muscle cellnot undergoing atrophy are harvested from an animal. In another aspect,the muscle cell undergoing atrophy is harvested while the animal is in astate of fasting and the muscle cell not undergoing atrophy is harvestedprior to the state of fasting. In yet another aspect, the muscle cellundergoing atrophy is harvested from an immobilized muscle and themuscle cell not undergoing atrophy is harvested from a mobile muscle. Inyet another aspect, the muscle cell undergoing atrophy is harvested froman animal with spinal cord injury and the muscle cell not undergoingatrophy is harvested from a muscle that has received electricalstimulation. In yet another aspect, the Muscle Atrophy Signature isdetermined by selecting mRNAs commonly up regulated or commonly downregulated between two or more of the Muscle Atrophy Signatures of themethods described herein.

In a further aspect, the invention relates to a method for inhibitingmuscle atrophy in a mammal, the method comprising administering to themammal a therapeutically effective amount of a compound of identifiedusing the method described above.

8. Non-Medical Uses

Also provided are the uses of the disclosed compounds and products aspharmacological tools in the development and standardization of in vitroand in vivo test systems for the evaluation of the effects of inhibitorsof muscle atrophy related activity in laboratory animals such as cats,dogs, rabbits, monkeys, rats, fish, birds, and mice, as part of thesearch for new therapeutic agents of promoting muscle health, promotingnormal muscle function, and/or promoting healthy aging muscles.

E. EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.), but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. or is at ambient temperature, and pressure is ator near atmospheric.

Certain materials, reagents and kits were obtained from specific vendorsas indicated below, and as appropriate the vendor catalog, part or othernumber specifying the item are indicated. Vendors indicated below are asfollows: “Ambion” is Ambion, a division of Life TechnologiesCorporation, Austin, Tex., USA; “Applied Biosystems” is AppliedBiosystems, a division of Life Technologies Corporation, Carlsbad,Calif., USA; “Boehringer Mannheim” is Boehringer Mannheim Corporatin,Indiapolis, Ind., USA; “CardinalHealth” is Cardinal Health, Inc.,Dublin, Ohio, USA; “Cell Signaling” is Cell Signaling Technology, Inc.,Beverly, Massachussetts, USA; “Columbus Inst” is Columbus InstrumentsInternational, Columbus, Ohio, USA; “Harlan” is Harlan Laboratories,Indianapolis, Ind., USA; “Instrumedics” is Instrumedics, Inc., Richmond,Ill., USA; “Invitrogen” is Invitrogen Corporation, Carlsbad, Calif.,USA; “Microm” is the Microm division (Walldorf, Germany) of ThermoFisher Scientific Inc., Rockford, Ill., USA; “Millipore” is MilliporeCorporation, Billerica, Massachussetts, USA; a division of Merck KGaA,Darmstadt, Germany; “Ortho” is Ortho Clinical Diagnostics, Rochester,N.Y., USA; “Pierce” is Pierce Biotechnology, Inc., Milwaukee, Wis., USA,a division of Thermo Fisher Scientific, Inc.; “R&D Systems” is R&DSystems Inc., Minneapolis, Minn., USA; “Roche Diagnostics” is RocheDiagnostics Corporation, Indianapolis, Ind., USA; “Sakura” is SakuraFinetek USA, Inc., Torrance, Calif., USA; “Santa Cruz” is Santa CruzBiotechnology, Inc., Santa Cruz, Calif., USA; and, “Sigma” isSigma-Aldrich Corporation, Saint Louis, Mo., USA.

1. General Methods

a. Human Subject Protocol.

The study referred to herein was approved by the Institutional ReviewBoard at the University of Iowa, and involved seven healthy adults whogave their informed consent before participating. One week prior to thefasting study, subjects made one visit to the Clinical Research Unit(“CRU”) for anthropometric measurements, a dietary interview thatestablished each subject's routine food intake and food preferences, andbaseline determinations of blood hemoglobin (“Hb”) A1c turbidimetricimmunoinhibition using the BM/Hitachi 911 analyzer (BoehringerMannheim); plasma triglycerides and plasma free T4 and TSH byelectrochemiluminescence immunoassay using the Elecsys® System (RocheDiagnostics); plasma CRP by immuno-turbidimetric assay using the RocheCobas Integra® high-sensitivity assay (Roche Diagnostics); and, plasmaTNF-α levels using the Quantikine® Kit (R&D Systems). To ensure thatsubjects were eating their routine diet prior to the fasting study,subjects ate only meals prepared by the CRU dietician (based on thedietary interview) for 48 hours before the fasting study. The fastingstudy began at t=0 hours, when subjects were admitted to the CRU andbegan fasting. While fasting, subjects remained in the CRU and wereencouraged to maintain their routine physical activities. Water wasallowed ad libitum, but caloric intake was not permitted. At about 40hours, a percutaneous biopsy was taken from the vastus lateralis muscleusing a Temno® Biopsy Needle (CardinalHealth; Cat # T1420) underultrasound guidance. Subjects then ate a CRU-prepared mixed meal, and att=46 hours, a muscle biopsy was taken from the contralateral vastuslateralis muscle. Plasma glucose and insulin levels were measured att=36, 40, 42 and 46 hours; the Elecsys® system was used to quantitateplasma insulin. Our study protocol of humans with spinal cord injury wasdescribed previously (Adams C M, et al. (2011) Muscle Nerve.43(1):65-75).

b. Microarray Analysis of Human Skeletal Muscle mRNA Levels.

Following harvest, skeletal muscle samples were immediately placed inRNAlater (Ambion) and stored at −80° C. until further use. Total RNA wasextracted using TRIzol solution (Invitrogen), and microarrayhybridizations were performed at the University of Iowa DNA Facility, asdescribed previously (Lamb J, et al. (2006) Science (New York, N.Y.313(5795):1929-1935). The log₂ hybridization signals as shown hereinreflect the mean signal intensity of all exon probes specific for anindividual mRNA. To determine which human skeletal muscle mRNAs weresignificantly altered by fasting (P≤0.02), paired t-tests were used tocompare fasted and fed log 2 signals. To determine which mouse skeletalmuscle mRNAs were significantly altered by ursolic acid (P≤0.005),unpaired t-tests were used to compare log₂ signals in mice fed controldiet or diet supplemented with ursolic acid. Highly expressed mRNAs weredefined as those significantly altered mRNAs that were repressed from orinduced to a log₂ signal>8. These raw microarray data from humans andmice have been deposited in NCBI's Gene Expression Omnibus (“GEO”) andare accessible through GEO Series accession numbers GSE28016 andGSE28017, respectively. Exon array studies of the effects of fasting onmouse skeletal muscle, and the effects of spinal cord injury on humanskeletal muscle were described previously (Adams C M, et al. (2011)Muscle & nerve 43(1):65-75; Ebert S M, et al. (2010) MolecularEndocrinology 24(4):790-799).

c. Quantitative Real-Time RT-PCR (qPCR).

TRIzol-extracted mRNA was treated with DNase I using the Turbo DNA-freekit (Ambion). qPCR analysis of human mRNA and mouse IGF-I mRNA wasperformed using TaqMan Gene Expression Assays (Applied Biosystems).First strand cDNA was synthesized from 2 μg of RNA using the HighCapacity cDNA Reverse Transcription Kit (Applied Biosystems, Part No.4368814). The real time PCR contained, in a final volume of 20 μl, 20 ngof reverse transcribed RNA, 1 μl of 20× TaqMan Gene Expression Assay,and 10 μl of TaqMan Fast Universal PCR Master Mix (Applied Biosystems;Part No. 4352042). qPCR was carried out using a 7500 Fast Real-Time PCRSystem (Applied Biosystems) in 9600 emulation mode. qPCR analysis ofmouse atrogin-1 and MuRF1 mRNA levels was performed as previouslydescribed (Ebert S M, et al. (2010) Molecular Endocrinology24(4):790-799). All qPCR reactions were performed in triplicate and thecycle threshold (Ct) values were averaged to give the final results. Toanalyze the data, the ΔCt method was used, with the level of 36B4 mRNAserving as the invariant control.

d. Mouse Protocols.

Male C57BL/6 mice, ages 6-8 weeks, were obtained from NCI, housed incolony cages with 12 h light/12 h dark cycles, and used for experimentswithin 3 weeks of their arrival. Unless otherwise indicated, mice weremaintained on standard chow (Harlan; Teklad Diet, Formula 7013, NIH-31Modified Open Formula Mouse/Rat Sterilizable Diet). Metformin (Sigma)was dissolved in 0.9% NaCl at a concentration of 250 mg/ml. Ursolic acid(Enzo Life Sciences) was dissolved in corn oil at a concentration of 200mg/ml (for i.p. injections); alternatively, the ursolic acid was addeddirectly to standard chow (Harlan; Teklad Diet, Formula 7013) orstandard high fat diet (Harlan; Teklad Diet, Formula TD.93075) as acustomized chow. Oleanolic acid (Sigma) was dissolved in corn oil at aconcentration of 200 mg/ml. Mice were fasted by removing food, but notwater, for 24 hours. Fasting blood glucose levels were obtained from thetail vein with an ACCU-CHEK® Aviva glucose meter (Roche Diagnostics).Unilateral hindlimb muscle denervation was performed by transsecting thesciatic nerve under anesthesia, and was followed by administration ofursolic acid (200 mg/kg) or vehicle alone (corn oil) via i.p injectiontwice daily for 7 days. Forelimb grip strength was determined using agrip strength meter equipped with a triangular pull bar (Columbus Inst).Each mouse was subjected to 5 consecutive tests to obtain the peakvalue. Plasma IGF-I and leptin levels were measured by RIA at theVanderbilt University Hormone Assay Core Facility. Plasma cholesterol,triglyceride, creatinine, bilirubin and ALT were measured using theVITROS® 350 Chemistry System (Ortho). All animal procedures wereapproved by the Institutional Animal Care and Use Committee of theUniversity of Iowa.

e. Histological Analysis.

Following harvest, tissues were immediately placed in isopentane thathad been chilled to −160° C. with liquid N₂. Muscles were embedded intissue freezing medium, and 10 μm sections from the mid-belly wereprepared using a Microm HM 505 E cryostat equipped with a CryoJanesectioning system (Instrumedics). Adipose tissue was fixed in 10%neutral buffered formalin, embedded in paraffin, and then 4 μm sectionswere prepared using a Microm HM355 S motorized microtome (Microm).Hematoxylin and eosin stains were performed using a DRS-601 automaticslide stainer (Sakura), and examined on an Olympus IX-71 microscopeequipped with a DP-70 camera. Image analysis was performed using ImageJsoftware (public domain, available from the National Institutes ofHealth, USA). Muscle fiber diameter was measured using the lesserdiameter method, as described elsewhere (Dubowitz V, et al. (2007)Muscle biopsy: a practical approach (Saunders Elsevier, Philadelphia)3rd Ed pp XIII, 611 s).

f. Analysis of IGF-I and Insulin-Mediated Protein Phosphorylation.

Mouse quadriceps muscles were snap frozen in liquid N₂, and Triton-X 100soluble protein extracts were prepared as described previously (Ebert SM, et al. (2010) Molecular endocrinology 24(4):790-799). Mouse C2C12myoblasts were obtained from American Type Culture Collection (“ATCC”),and maintained in Dulbecco's modified Eagle's medium (DMEM; ATCC#30-2002) containing antibiotics (100 units/ml penicillin, 100 μg/mlstreptomycin sulfate) and 10% (v/v) fetal bovine serum (FBS). On day 0,myotubes were set-up in 6-well plates at a density of 2.5×10⁵cells/well. On day 2, differentiation into myotubes was induced byreplacing 10% FBS with 2% horse serum. On day 7, myotubes wereserum-starved by washing 2 times with phosphate buffered saline, andthen adding fresh serum-free media. After 16 hours of serum-starvation,10 μM ursolic acid (from a 10 mM stock prepared in DMSO), or an equalvolume of DMSO, with or without 10 nM mouse IGF-I (Sigma; Cat. No.18779) or 10 nM bovine insulin (Sigma; Cat. No. 16634) was directlyadded to the media. For analysis of Akt, S6K, ERK and FoxOphosphorylation, myotubes were incubated in the presence or absence ofursolic acid, IGF-I and/or insulin for 20 min, and then harvested intoSDS lysis buffer (10 mM Tris-HCl, pH 7.6, 100 mM NaCl, 1% (w/v) SDS, 1μg/ml pepstatin A, 2 μg/ml aprotonin, 10 μg/ml leupeptin, 200 μMphenylmethylsulfonyl fluoride and a 1:100 dilution of phosphataseinhibitor cocktail 3 (Sigma). An aliquot of each muscle extract or celllysate was mixed with 0.25 volume of sample buffer (250 mM Tris-HCl, pH6.8, 10% SDS, 25% glycerol, 0.2% (w/v) bromophenol blue, and 5% (w/v)2-mercaptoethanol) and heated for 5 min at 95° C., whereas a separatealiquot was used to determine protein concentration by the BCA kit(Pierce). Samples (25 μg) were subjected to 8% SDS-PAGE, thentransferred to Hybond-C extra nitrocellulose filters (Millipore).Immunoblots were performed at 4° C. for 16 h using a 1:2000 dilution ofantibodies detecting total Akt, phospho-Akt(Ser473), total S6K,phospho-S6K(T421/S424), total ERK1/2, phospho-ERK(T202/Y204), FoxO3a, orphospho-FoxO1(T24)/FoxO3a(T32) (Cell Signaling). For analysis of IGF-1receptor or insulin receptor phosphorylation, myotubes were incubated inthe presence or absence of ursolic acid, IGF-I and/or insulin for 2 min,and then harvested into RIPA buffer (10 mM Tris-HCL, pH 7.4, 150 mMNaCl, 0.1% (w/v) SDS, 1% (w/v) Triton X-100, 1% Na deoxycholate, 5 mMEDTA, 1 mM NaF, 1 mM Na orthovanadate, 1 μg/ml pepstatin A, 2 μg/mlaprotonin, 10 μug/ml leupeptin, 200 μM phenylmethylsulfonyl fluoride,1:100 dilution of phosphatase inhibitor cocktail 2 (Sigma) and a 1:100dilution of phosphatase inhibitor cocktail 3 (Sigma). The proteinconcentration was measured using the BCA kit, after which the extractwas diluted to a concentration of 1 mg/ml in RIPA buffer (final volume500 μl). Then 2 μg anti-IGF-1 receptor β antibody (Cell Signaling) or 2μg anti-insulin receptor β antibody (Santa Cruz) was added with 50 μlprotein G plus Sepharose beads (Santa Cruz), and then the samples wererotated at 4° C. for 16 h. Immunoprecipitates were washed three timesfor 20 min with 1 ml RIPA buffer and then mixed with 100 μl samplebuffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 5% glycerol, 0.04% (w/v)bromophenol blue and 5% (w/v) 2-mercaptoethanol), then boiled for 5 min.Immunoprecipitates were subjected to 8% SDS-PAGE. For analysis of totalIGF-1 receptor, phospho-insulin receptor and total insulin receptor,proteins were transferred to Hybond-C extra nitrocellulose filters(Millipore). For analysis of phospho-IGF-1 receptor, proteins weretransferred to PVDF membranes (Bio-Rad). Immunoblots were performed atroom temperature using a 1:2000 dilution of anti-IGF-1 receptor βantibody, 1:5000 dilution of mouse anti-phospho-tyrosine 4G10 monoclonalantibody (Millipore), a 1:2000 dilution of anti-insulin receptor β, or1:2000 dilution of anti-phospho-insulin receptor β (Y1162/1163) (SantaCruz).

g. PTP1B Inhibition Via RNA Interference.

The plasmids pCMV-miR-PTP1B #1 and pCMV-miR-PTP1B #2 were generated byligating PTPN1-specific oligonucleotide duplexes (Invitrogen) into thepcDNA6.2GW/EmGFP miR plasmid (Invitrogen), which contains a CMV promoterdriving co-cistronic expression of engineered pre-miRNAs and EmGFP.pCMV-miR-control encodes a non-targeting pre-miRNA hairpin sequence(miR-neg control; Invitrogen) in pcDNA6.2GW/EmGFP miR plasmid. MaleC57BL/6 mice were obtained from NCI at ages 6-8 weeks, and used forexperiments within 3 weeks of their arrival. Electroporation of mousetibialis anterior muscles and isolation of skeletal muscle RNA wasperformed as described previously (Ebert S M, et al. (2010) Molecularendocrinology 24(4):790-799). First strand cDNA was synthesized in a 20μl reaction that contained 2 μg of RNA, random hexamer primers andcomponents of the High Capacity cDNA reverse transcription kit (AppliedBiosystems). qPCR analysis of PTPN1 mRNA levels was performed using aTaqman expression assay as described previously (Ebert S M, et al.(2010) Molecular endocrinology 24(4):790-799). qPCR was carried outusing a 7500 Fast Real-Time PCR System (Applied Biosystems). All qPCRreactions were performed in triplicate and the cycle threshold (Ct)values were averaged to give the final results. Fold changes weredetermined by the ΔCt method, with level of 36B4 mRNA serving as theinvariant control. Skeletal muscle sections were prepared andtransfected (EmGFP-positive) muscle fibers were identified and measuredas described previously (Ebert S M, et al. (2010) Molecularendocrinology 24(4):790-799).

h. Measurement of Serum Ursolic Acid Levels.

Ursolic acid is extracted from serum using a 10:1 mixture ofhexane:propanol (recovery>90%), and then conjugated via its carboxylicacid group to 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate(Invitrogen; Ne-OTf), a moiety that enhances TUV and fluorescencedetection. Derivatized samples are then analyzed on a Waters AcquityUPLC equipped with a 100×2.1 mm C18 HSS column with 1.8 μm beads (WatersPart No. 186003533) and a TUV detector.

2. Identification of Therapeutics to Treat Muscle Atrophy

Skeletal muscle atrophy is common and debilitating condition that lacksa pharmacologic therapy. To identify and develop new therapeuticapproaches to this pathophysiological condition (FIG. 1), an approachusing gene expression signatures to connect small molecules, genes, anddisease was used. Briefly, 63 mRNAs were identified that were regulatedby fasting in both human and mouse muscle, and 29 mRNAs that wereregulated by both fasting and spinal cord injury in human muscle. Thesetwo unbiased mRNA expression signatures of muscle atrophy were used toquery the Connectivity Map, an algorithm that allows gene signaturedatasets to be used to find relationships between small molecules,genes, and disease.

Three complimentary studies to characterize global atrophy-associatedchanges in skeletal muscle mRNA levels in humans and mice were carriedout. These three studies determined the effects of: A) fasting on humanskeletal muscle mRNA levels as described herein, B) spinal cord injury(“SCI”) on human skeletal mRNA levels (Adams C M, et al. (2011) Muscle &nerve 43(1):65-75) and C) fasting on mouse skeletal muscle mRNA levels(Ebert S M, et al. (2010) Molecular endocrinology 24(4):790-799). Ineach study, exon expression arrays were used to quantitate levels ofmore than 16,000 mRNAs. Although there were many significant changes ineach study, analysis focused on mRNAs whose levels were similarlyaltered in at least two atrophy models. Thus, by comparing the effectsof fasting on human and mouse skeletal muscle, there were two sets ofmRNAs identified: a) 31 mRNAs that were increased by fasting in bothspecies, and b) 32 mRNAs that were decreased by fasting in both species.These evolutionarily conserved, fasting-regulated skeletal muscle mRNAswere termed “muscle atrophy signature-1” (see FIG. 2). Next, the effectsof fasting and SCI on human skeletal muscle were determined and two setsof mRNAs were identified: a) 18 mRNAs that were increased by fasting andSCI, and b) 17 mRNAs that were decreased by fasting and SCI. This secondgroup of mRNAs was termed “muscle atrophy signature-2” (see FIG. 3).Almost all of the mRNAs in muscle atrophy signatures-1 and -2 havepreviously uncharacterized roles in normal or atrophied skeletal muscle.It was next hypothesized that pharmacologic compounds whose effects oncellular mRNA levels were opposite to muscle atrophy signatures-1 and -2might inhibit skeletal muscle atrophy. To identify candidate compounds,the Connectivity Map (Lamb J, et al. (2006) Science (New York, N.Y.313(5795):1929-1935) was used to compare muscle atrophy signatures-1 and-2 to mRNA expression signatures of >1300 bioactive small molecules.These results identified several predicted inhibitors of human skeletalmuscle atrophy, including ursolic acid. The predicted inhibitors ofhuman skeletal muscle atrophy, i.e. compounds with negative connectivitywith the muscle atrophy signatures, are shown in Tables 2 and 3 below.Table 2 shows compounds with negative connectivity to human muscleatrophy signature-1 (see FIG. 2 for mRNAs in the signature), whereasTable 3 shows compounds with negative connectivity to human muscleatrophy signature-2 (see FIG. 3 for mRNAs in the signature).

As a proof-of-concept of the utility of muscle atrophy signatures-1 and-2 described herein, the effects of ursolic acid were assessed in mice,and surprisingly it was discovered ursolic acid inhibited muscle atrophyand promoted muscle hypertrophy.

TABLE 2 Compounds with negative connectivity to human muscle atrophysignature-1. Cmap name/cell Connectivity % Non- line score n Enrichmentp Specificity null conessine - HL60 −0.752 1 −0.991 — — 100 allantoin -HL60 −0.622 1 −0.954 — — 100 conessine - PC3 −0.598 1 −0.941 — — 100tacrine - HL60 −0.551 1 −0.91 — — 100 tomatidine - HL60 −0.497 1 −0.873— — 100 tomatidine - PC3 −0.483 1 −0.861 — — 100 naringenin - PC3 −0.4621 −0.846 — — 100 allantoin - MCF7 −0.347 2 −0.735 0.13873 0.1118 50tomatidine - MCF7 −0.343 2 −0.78 0.09489 0.2263 50 naringenin - MCF7−0.219 2 −0.546 0.4127 0.6589 50 allantoin - PC3 −0.077 2 −0.414 0.784460.7654 50

TABLE 3 Compounds with negative connectivity to human muscle atrophysignature-2. Connectivity % Non- Cmap name/cell line score n Enrichmentp Specificity null tacrine - HL60 −0.870 1 −0.998 — — 100 tomatidine -PC3 −0.861 1 −0.998 — — 100 naringenin - PC3 −0.754 1 −0.990 — — 100betulinic acid - HL60 −0.569 1 −0.929 — — 100 conessine - HL60 −0.543 1−0.915 — — 100 allantoin - MCF7 −0.486 2 −0.840 0.05114 0.04710 100naringenin - MCF7 −0.314 2 −0.460 0.64871 0.84500 50 tomatidine - MCF7−0.281 2 −0.611 0.30586 0.65260 50

3. Effects of Fasting on Skeletal Muscle mRNA Expression in Humans.

Prolonged fasting induces muscle atrophy, but its effects on global mRNAexpression in human skeletal muscle were not known heretofore. In orderto determine the relationship between global mRNA expression and humanskeletal muscle status, seven healthy adult human volunteers (3 male and4 female) with ages ranging from 25 to 69 years (mean=46 years) werestudied. The overall study design is shown in FIG. 4A. The mean bodymass index of these subjects (±SEM) was 25±1. Their mean weight was69.4±4.8 kg. Baseline circulating levels of hemoglobin A1c (HbA1c),triglycerides (TG), thyroid-stimulating hormone (TSH), free thyroxine(free T4), C-reactive protein (CRP) and tumor necrosis factor-α (TNF-α)were within normal limits (FIG. 4A). The table (FIG. 4A, insert) showsbaseline circulating metabolic and inflammatory markers. The graph showsplasma glucose and insulin levels (FIG. 4A). Data are means±SEM from theseven study subjects. In some cases, the error bars are too small tosee. While staying in the University of Iowa Clinical Research Unit, thesubjects fasted for 40 h by forgoing food but not water. The mean weightloss during the fast was 1.7±0.1 kg (3±0% of the initial body weight).

After the 40 h fast, a muscle biopsy was obtained from the subjects'vastus lateralis (VL) muscle. Immediately after the muscle biopsy, thesubjects ate a mixed meal. Five hours later (six hours after the firstbiopsy), a second muscle biopsy from their contralateral VL muscle.Thus, each subject had a muscle biopsy under fasting and nonfastingconditions. As expected, plasma glucose and insulin levels were low atthe end of the 40 h fast, rose after the meal, and returned to baselineby the time of the second biopsy (FIG. 4A). These data indicatecomparable levels of plasma glucose and insulin at the times of thefirst (fasting) and second (nonfasting) muscle biopsies.

To determine the effect of fasting on skeletal muscle mRNA expression,RNA was isolated from the paired muscle biopsies and then analyzed itwith exon expression arrays. Using P≤0.02 (by paired t-test) as criteriafor statistical significance, it was found that 281 mRNAs were higher inthe fasting state and 277 were lower (out of >17,000 mRNAs measured; seeFIG. 4B). A complete list of these fasting-responsive mRNAs is shownbelow in Table X1 (“Change” is the mean log 2 change or differencebetween fasting and fed states). The data in Table X1 is for all mRNAsin this study whose levels were increased or decreased by fasting(P≤0.02 by paired t-test).

Representative fasting-responsive human skeletal muscle mRNAs, and theeffect of fasting on their log 2 hybridization signals, as assessed byAffymetrix Human Exon 1.0 ST arrays are shown in FIG. 4B. In eachsubject, the fasting signal was normalized to the nonfasting signal fromthe same subject. Data are means±SEM from 7 subjects. P≤0.02 by pairedt-test for all mRNAs shown. The complete set of 458 fasting-responsivemRNAs is shown in Table X1. Most of the differentially expressed mRNAsidentified as altered by fasting surprisingly did not have previouslyknown roles in muscle atrophy. However, fasting increased several mRNAsthat encode proteins with known roles in catabolic processes such as fatoxidation, reverse cholesterol transport, thermogenesis, inhibition ofprotein synthesis, autophagy, ubiquitin-mediated proteolysis, glutaminetransport and heme catabolism (FIG. 4B). Of these, atrogin-1, MuRF1 andZFAND5 mRNAs encode proteins known to be required for skeletal muscleatrophy in mice (Bodine S C, et al. (2001) Science (New York, N.Y.294(5547): 1704-1708; Hishiya A, et al. (2006) The EMBO journal25(3):554-564). Conversely, fasting significantly decreased severalmRNAs encoding proteins with known roles in anabolic processes such asglycogen synthesis, lipid synthesis and uptake, polyamine synthesis,iron uptake, angiogenesis, and mitochondrial biogenesis (FIG. 4B). Ofthese, PGC-1α mRNA encodes a protein that inhibits atrophy-associatedgene expression and skeletal muscle atrophy in mice (Sandri M, et al.(2006) Proceedings of the National Academy of Sciences of the UnitedStates of America 103(44): 16260-16265).

The results were further validated using qPCR to analyze RNA from pairedfed and fasted skeletal muscle biopsy samples obtained from sevenhealthy human subjects (see FIG. 5; data are means±SEM; * P≤0.01 bypaired t-test.). In each subject, the fasting mRNA level was normalizedto the nonfasting level, which was set at 1. The mRNA encoding myostatin(MSTN) is a control transcript whose level was not altered by fasting,as assessed by exon expression arrays. Taken together, these dataestablished an mRNA expression signature of fasting in human skeletalmuscle.

TABLE X1 Fasting-responsive human mRNAs. Change Affymetrix Accession(Fasting- ID mRNA Gene Assignment No. Fed) SEM P 3062082 PDK4 NM_002612// NM_002612 2.15 0.34 0.000 PDK4 // pyruvate dehydrogenase kinase,isozyme 4 // 7q21.3 // 5166 2319340 SLC25A33 NM_032315 // NM_032315 1.420.41 0.007 SLC25A33 // solute carrier family 25, member 33 // 1p36.22 //84275 3165957 IFNK NM_020124 // NM_020124 0.96 0.28 0.007 IFNK //interferon, kappa //—// 56832 /// ENST00000276943 // IF 3424158 MYF6NM_002469 // NM_002469 0.95 0.12 0.000 MYF6 // myogenic factor 6(herculin) // 12q21 // 4618 /// ENST00000 3422144 LGR5 NM_003667 //NM_003667 0.88 0.12 0.000 LGR5 // leucine- rich repeat- containing Gprotein-coupled receptor 5 2356115 TXNIP NM_006472 // NM_006472 0.850.22 0.004 TXNIP // thioredoxin interacting protein // 1q21.1 // 10628/// ENS 3233605 PFKFB3 NM_004566 // NM_004566 0.84 0.18 0.002 PFKFB3 //6- phosphofructo-2- kinase/fructose-2,6- biphosphatase 3 // 3151607FBXO32 NM_058229 // NM_058229 0.82 0.19 0.002 FBXO32 // F-box protein 32// 8q24.13 // 114907 /// NM_148177 // FB 2745547 GAB1 NM_207123 //NM_207123 0.71 0.08 0.000 GAB1 // GRB2- associated binding protein 1 //4q31.21 // 2549 /// NM 3173479 FOXD4L3 NM_199135 // NM_199135 0.68 0.250.017 FOXD4L3 // forkhead box D4- like 3 // 9q13 // 286380 /// NM_012184/ 3199500 CER1 NM_005454 // NM_005454 0.64 0.24 0.019 CER1 // cerberus1, cysteine knot superfamily, homolog (Xenopuslae 3444309 TAS2R9NM_023917 // NM_023917 0.63 0.22 0.015 TAS2R9 // taste receptor, type 2,member 9 // 12p13 // 50835 /// EN 3452323 SLC38A2 NM_018976 // NM_0189760.62 0.13 0.001 SLC38A2 // solute carrier family 38, member 2 // 12q //54407 /// E 3381843 UCP3 NM_003356 // NM_003356 0.59 0.04 0.000 UCP3 //uncoupling protein 3 (mitochondrial, proton carrier) // 11q 3147508KLF10 NM_005655 // NM_005655 0.58 0.11 0.001 KLF10 // Kruppel- likefactor 10 // 8q22.2 // 7071 /// NM_001032282 3982534 LPAR4 NM_005296 //NM_005296 0.57 0.17 0.008 LPAR4 // lysophosphatidic acid receptor 4 //Xq13-q21.1 // 2846 /// 3384321 RAB30 NM_014488 // NM_014488 0.56 0.210.019 RAB30 // RAB30, member RAS oncogene family // 11q12-q14 // 27314// 3256192 C10orf116 NM_006829 // NM_006829 0.55 0.19 0.013 C10orf116 //chromosome 10 open reading frame 116 // 10q23.2 // 109 2705690 GHSRNM_198407 // NM_198407 0.54 0.20 0.016 GHSR // growth hormonesecretagogue receptor // 3q26.31 // 2693 /// 3326938 LOC100130104AF274942 // AF274942 0.53 0.16 0.009 LOC100130104 // PNAS-17 // 11p13 //100130104 2318656 PER3 NM_016831 // NM_016831 0.52 0.16 0.009 PER3 //period homolog 3 (Drosophila) // 1p36.23 // 8863 /// ENST00 3209623ZFAND5 NM_001102420 // NM_001102420 0.51 0.13 0.005 ZFAND5 // zincfinger, AN1-type domain 5 // 9q13-q21 // 7763 /// 3741300 OR1D4NM_003552 // NM_003552 0.50 0.19 0.019 OR1D4 // olfactory receptor,family 1, subfamily D, member 4 // 17p 2899176 HIST1H2BD NM_138720 //NM_138720 0.49 0.16 0.010 HIST1H2BD // histone cluster 1, H2bd // 6p21.3// 3017 /// NM_02106 3439256 RPS11 ENST00000270625 ENST00000270625 0.490.11 0.002 // RPS11 // ribosomal protein S11 // 19q13.3 // 6205 ///BC10002 2973232 KIAA0408 NM_014702 // NM_014702 0.49 0.14 0.006 KIAA0408// KIAA0408 // 6q22.33 // 9729 /// NM_001012279 // C6orf17 3291151RHOBTB1 NM_014836 // NM_014836 0.48 0.09 0.001 RHOBTB1 // Rho- relatedBTB domain containing 1 // 10q21.2 // 9886/ 2358136 C1orf51 BC027999 //BC027999 0.48 0.17 0.016 C1orf51 // chromosome 1 open reading frame 51// 1q21.2 // 148523 // 3948936 — — 0.47 0.18 0.020 3944129 HMOX1NM_002133 // NM_002133 0.46 0.13 0.006 HMOX1 // hemeoxygenase(decycling) 1 // 22q12|22q13.1 // 3162 /// 2968652 SESN1 NM_014454 //NM_014454 0.46 0.12 0.004 SESN1 // sestrin 1 // 6q21 // 27244 ///ENST00000302071 // SESN1 // 2951881 PXT1 NM_152990 // NM_152990 0.450.14 0.008 PXT1 // peroxisomal, testis specific 1 // 6p21.31 // 222659/// ENS 2819747 POLR3G NM_006467 // NM_006467 0.45 0.13 0.007 POLR3G //polymerase (RNA) III (DNA directed) polypeptide G (32 kD) 2957384 GSTA2NM_000846 // NM_000846 0.44 0.10 0.002 GSTA2 // glutathione S-transferase A2 // 6p12.1 // 2939 /// NM_1536 4014387 RPSA NM_002295 //NM_002295 0.44 0.16 0.018 RPSA // ribosomal protein SA // 3p22.2 // 3921/// NM_001012321 // 3021158 C7orf58 NM_024913 // NM_024913 0.44 0.070.000 C7orf58 // chromosome 7 open reading frame 58 // 7q31.31 // 79974/ 2976155 OLIG3 NM_175747 // NM_175747 0.44 0.12 0.006 OLIG3 //oligodendrocyte transcription factor 3 // 6q23.3 // 167826 3261886C10orf26 NM_017787 // NM_017787 0.44 0.17 0.019 C10orf26 // chromosome10 open reading frame 26 // 10q24.32 // 5483 2489169 — — 0.42 0.12 0.0062790062 TMEM154 NM_152680 // NM_152680 0.42 0.14 0.012 TMEM154 //transmembrane protein 154 // 4q31.3 // 201799 /// ENST00 3792656CCDC102B NM_024781 // NM_024781 0.42 0.12 0.007 CCDC102B // coiled-coildomain containing 102B // 18q22.1 // 79839 3554282 INF2 NM_022489 //NM_022489 0.41 0.14 0.012 INF2 // inverted formin, FH2 and WH2 domaincontaining // 14q32.33 2614142 NR1D2 NM_005126 // NM_005126 0.39 0.150.019 NR1D2 // nuclear receptor subfamily 1, group D, member 2 // 3p24.23404636 GABARAPL1 NM_031412 // NM_031412 0.39 0.10 0.004 GABARAPL1 //GABA(A) receptor- associated protein like 1 // 12p13.2 3063856tcag7.1177 ENST00000292369 ENST00000292369 0.39 0.09 0.003 // tcag7.1177// opposite strand transcription unit to STAG3 // 3461981 TSPAN8NM_004616 // NM_004616 0.39 0.14 0.015 TSPAN8 // tetraspanin 8 //12q14.1-q21.1 // 7103 /// ENST0000039333 2908154 C6orf206 BC029519 //BC029519 0.39 0.09 0.003 C6orf206 // chromosome 6 open reading frame 206// 6p21.1 // 221421 3415046 FLJ33996 AK091315 // AK091315 0.39 0.150.019 FLJ33996 // hypothetical protein FLJ33996 // 12q13.13 // 283401/// 3326400 CAT NM_001752 // NM_001752 0.39 0.09 0.003 CAT // catalase// 11p13 // 847 /// ENST00000241052 // CAT // catal 2390322 OR2M5NM_001004690 // NM_001004690 0.38 0.12 0.011 OR2M5 // olfactoryreceptor, family 2, subfamily M, member 5 // 2402536 TRIM63 NM_032588 //NM_032588 0.38 0.12 0.009 TRIM63 // tripartite motif-containing 63 //1p34-p33 // 84676 /// E 2976768 CITED2 NM_006079 // NM_006079 0.37 0.100.005 CITED2 // Cbp/p300- interacting transactivator, with Glu/Asp-richca 3218528 ABCA1 NM_005502 // NM_005502 0.37 0.14 0.016 ABCA1 // ATP-binding cassette, sub-family A (ABC1), member 1 // 9q3 3377861DKFZp761E198 NM_138368 // NM_138368 0.37 0.06 0.000 DKFZp761E198 //DKFZp761E198 protein // 11q13.1 // 91056 /// BC1091 2961347 FILIP1NM_015687 // NM_015687 0.37 0.10 0.005 FILIP1 // filamin A interactingprotein 1 // 6q14.1 // 27145 /// EN 3097580 C8orf22 NM_001007176 //NM_001007176 0.37 0.08 0.002 C8orf22 // chromosome 8 open reading frame22 // 8q11 // 492307 3755655 FBXL20 NM_032875 // NM_032875 0.35 0.080.002 FBXL20 // F-box and leucine-rich repeat protein 20 // 17q12 //8496 3057505 CCL26 NM_006072 // NM_006072 0.35 0.12 0.012 CCL26 //chemokine (C-C motif) ligand 26 // 7q11.23 // 10344 /// EN 3307795C10orf118 NM_018017 // NM_018017 0.35 0.13 0.020 C10orf118 // chromosome10 open reading frame 118 // 10q25.3 // 550 3654699 NUPR1 NM_001042483// NM_001042483 0.35 0.10 0.007 NUPR1 // nuclear protein 1 // 16p11.2 //26471 /// NM_012385 // 3778252 ANKRD12 NM_015208 // NM_015208 0.34 0.080.002 ANKRD12 // ankyrin repeat domain 12 // 18p11.22 // 23253 ///NM_001 2662560 C3orf24 NM_173472 // NM_173472 0.34 0.08 0.002 C3orf24 //chromosome 3 open reading frame 24 // 3p25.3 // 115795 / 3896370 RP5-NM_019593 // NM_019593 0.34 0.10 0.007 1022P6.2 RP5-1022P6.2 //hypothetical protein KIAA1434 // 20p12.3 // 56261 / 3389566 KBTBD3NM_198439 // NM_198439 0.34 0.08 0.003 KBTBD3 // kelch repeat and BTB(POZ) domain containing 3 // 11q22.3 3247818 FAM133B NM_152789 //NM_152789 0.34 0.11 0.010 FAM133B // family with sequence similarity133, member B // 7q21.2 2457988 ZNF706 AF275802 // AF275802 0.34 0.120.016 ZNF706 // zinc finger protein 706 // 8q22.3 // 51123 /// BC015925// 3525234 IRS2 NM_003749 // NM_003749 0.34 0.09 0.004 IRS2 // insulinreceptor substrate 2 // 13q34 // 8660 /// ENST00000 2730281 ODAMNM_017855 // NM_017855 0.34 0.12 0.016 ODAM // odontogenic, ameloblastasssociated // 4q13.3 // 54959 /// 3768969 ABCA5 NM_018672 // NM_0186720.33 0.10 0.008 ABCA5 // ATP- binding cassette, sub-family A (ABC1),member 5 // 17q 3687494 MAPK3 NM_001040056 // NM_001040056 0.33 0.090.004 MAPK3 // mitogen- activated protein kinase 3 // 16p11.2 // 5595 /3405396 CREBL2 NM_001310 // NM_001310 0.33 0.07 0.002 CREBL2 // cAMPresponsive element binding protein-like 2 // 12p13 / 3647504 PMM2NM_000303 // NM_000303 0.33 0.10 0.008 PMM2 // phosphomannomutase 2 //16p13.3-p13.2 // 5373 /// ENST00000 3392840 BUD13 NM_032725 // NM_0327250.33 0.07 0.002 BUD13 // BUD13 homolog (S. cerevisiae) // 11q23.3 //84811 /// ENST 3453837 TUBA1A NM_006009 // NM_006009 0.33 0.07 0.002TUBA1A // tubulin, alpha 1a // 12q12-q14.3 // 7846 /// ENST000003012409310 ELOVL1 NM_022821 // NM_022821 0.32 0.09 0.005 ELOVL1 //elongation of very long chain fatty acids (FEN1/Elo2, SUR 3837707 ZNF114NM_153608 // NM_153608 0.31 0.09 0.007 ZNF114 // zinc finger protein 114// 19q13.32 // 163071 /// ENST000 3504434 XPO4 NM_022459 // NM_0224590.31 0.10 0.009 XPO4 // exportin 4 // 13q11 // 64328 /// ENST00000255305// XPO4 // 2431877 — — 0.31 0.11 0.017 3837836 PSCD2 NM_017457 //NM_017457 0.31 0.05 0.000 PSCD2 // pleckstrin homology, Sec7 andcoiled-coil domains 2 (cytoh 3869396 ZNF432 NM_014650 // NM_014650 0.310.09 0.006 ZNF432 // zinc finger protein 432 // 19q13.33 // 9668 ///ENST00000 3981120 OGT NM_181672 // NM_181672 0.31 0.10 0.013 OGT //O-linked N- acetylglucosamine (GlcNAc) transferase (UDP- N-ace 2622607SLC38A3 NM_006841 // NM_006841 0.30 0.11 0.016 SLC38A3 // solute carrierfamily 38, member 3 // 3p21.3 // 10991 // 3978812 FOXR2 NM_198451 //NM_198451 0.30 0.09 0.008 FOXR2 // forkhead box R2 // Xp11.21 // 139628/// ENST00000339140 / 3571904 NPC2 NM_006432 // NM_006432 0.30 0.100.011 NPC2 // Niemann- Pick disease, type C2 // 14q24.3 // 10577 ///NM_00 2417945 PTGER3 NM_198715 // NM_198715 0.30 0.11 0.017 PTGER3 //prostaglandin E receptor 3 (subtype EP3) // 1p31.2 // 573 3059393 SEMA3ENM_012431 // NM_012431 0.30 0.09 0.009 SEMA3E // sema domain,immunoglobulin domain (Ig), short basic doma 2336456 MGC52498NM_001042693 // NM_001042693 0.30 0.10 0.011 MGC52498 // hypotheticalprotein MGC52498 // 1p32.3 // 348378 // 3726772 CROP NM_016424 //NM_016424 0.30 0.11 0.016 CROP // cisplatin resistance- associatedoverexpressed protein // 17 2784265 IL2 NM_000586 // IL2 NM_000586 0.290.11 0.019 // interleukin 2 // 4q26-q27 // 3558 /// ENST00000226730 //IL2 2495782 LIPT1 NM_145197 // NM_145197 0.29 0.10 0.012 LIPT1 //lipoyltransferase 1 // 2q11.2 // 51601 /// NM_145198 // LI 2377094PFKFB2 NM_006212 // NM_006212 0.29 0.10 0.012 PFKFB2 // 6-phosphofructo-2- kinase/fructose-2,6- biphosphatase 2 // 2469213 KLF11NM_003597 // NM_003597 0.29 0.10 0.011 KLF11 // Kruppel- like factor 11// 2p25 // 8462 /// ENST00000305883 3662387 HERPUD1 NM_014685 //NM_014685 0.29 0.07 0.003 HERPUD1 // homocysteine- inducible,endoplasmic reticulum stress-ind 3771215 ACOX1 NM_004035 // NM_0040350.29 0.10 0.013 ACOX1 // acyl- Coenzyme A oxidase 1, palmitoyl //17q24-q25| 17q25.1 3203135 TOPORS NM_005802 // NM_005802 0.28 0.11 0.018TOPORS // topoisomerase I binding, arginine/serine-rich // 9p21 //2805482 — — 0.28 0.09 0.008 3247757 UBE2D1 NM_003338 // NM_003338 0.280.08 0.007 UBE2D1 // ubiquitin- conjugating enzyme E2D 1 (UBC4/5homolog, yeast 3444147 KLRC1 NM_002259 // NM_002259 0.28 0.10 0.015KLRC1 // killer cell lectin-like receptor subfamily C, member 1 //3348891 C11orf57 NM_018195 // NM_018195 0.28 0.09 0.011 C11orf57 //chromosome 11 open reading frame 57 // 11q23.1 // 55216 3906942 SERINC3NM_006811 // NM_006811 0.28 0.07 0.003 SERINC3 // serine incorporator 3// 20q13.1-q13.3 // 10955 /// NM_1 2930418 UST NM_005715 // USTNM_005715 0.28 0.06 0.002 // uronyl-2- sulfotransferase // 6q25.1 //10090 /// ENST0000036 3188200 OR1L1 NM_001005236 // NM_001005236 0.280.09 0.011 OR1L1 // olfactory receptor, family 1, subfamily L, member 1// 3856075 ZNF682 NM_033196 // NM_033196 0.28 0.10 0.017 ZNF682 // zincfinger protein 682 // 19p12 // 91120 /// NM_00107734 3385951 NOX4NM_016931 // NM_016931 0.28 0.06 0.002 NOX4 // NADPH oxidase 4 //11q14.2-q21 // 50507 /// ENST00000263317 3523881 KDELC1 NM_024089 //NM_024089 0.28 0.06 0.002 KDELC1 // KDEL (Lys-Asp-Glu-Leu) containing 1// 13q33 // 79070 /// 2632778 EPHA6 NM_001080448 // NM_001080448 0.280.09 0.010 EPHA6 // EPH receptor A6 // 3q11.2 // 285220 ///ENST00000389672 3373272 OR5W2 NM_001001960 // NM_001001960 0.28 0.100.015 OR5W2 // olfactory receptor, family 5, subfamily W, member 2 //4017694 IRS4 NM_003604 // NM_003604 0.28 0.10 0.016 IRS4 // insulinreceptor substrate 4 // Xq22.3 // 8471 /// ENST0000 3545311 KIAA1737NM_033426 // NM_033426 0.28 0.07 0.003 KIAA1737 // KIAA1737 // 14q24.3// 85457 /// ENST00000361786 // KIA 3753860 CCL5 NM_002985 // NM_0029850.28 0.05 0.001 CCL5 // chemokine (C-C motif) ligand 5 // 17q11.2-q12 //6352 /// E 3617312 SLC12A6 NM_001042496 // NM_001042496 0.27 0.07 0.005SLC12A6 // solute carrier family 12 (potassium/chloride transpor 3351315UBE4A NM_004788 // NM_004788 0.27 0.07 0.004 UBE4A // ubiquitinationfactor E4A (UFD2 homolog, yeast) // 11q23.3 3755396 CCDC49 NM_017748 //NM_017748 0.27 0.09 0.013 CCDC49 // coiled- coil domain containing 49 //17q12 // 54883 /// EN 2870889 C5orf13 NM_004772 // NM_004772 0.27 0.090.010 C5orf13 // chromosome 5 open reading frame 13 // 5q22.1 // 9315/// 2775259 RASGEF1B NM_152545 // NM_152545 0.27 0.10 0.015 RASGEF1B //RasGEF domain family, member 1B // 4q21.21-q21.22 // 15 3165624 — — 0.270.06 0.003 2771654 CENPC1 NM_001812 // NM_001812 0.27 0.09 0.013 CENPC1// centromere protein C 1 // 4q12-q13.3 // 1060 /// ENST0000 3784670C18orf21 NM_031446 // NM_031446 0.27 0.08 0.008 C18orf21 // chromosome18 open reading frame 21 // 18q12.2 // 83608 2364231 DDR2 NM_001014796// NM_001014796 0.26 0.10 0.018 DDR2 // discoidin domain receptortyrosine kinase 2 // 1q23.3 // 3921442 SH3BGR NM_007341 // NM_0073410.26 0.08 0.007 SH3BGR // SH3 domain binding glutamic acid-rich protein// 21q22.3 2627368 C3orf49 BC015210 // BC015210 0.26 0.06 0.003 C3orf49// chromosome 3 open reading frame 49 // 3p14.1 // 132200 3250699EIF4EBP2 NM_004096 // NM_004096 0.26 0.10 0.018 EIF4EBP2 // eukaryotictranslation initiation factor 4E binding pro 3237788 PLXDC2 NM_032812 //NM_032812 0.26 0.09 0.013 PLXDC2 // plexin domain containing 2 //10p12.32-p12.31 // 84898 // 3285926 ZNF33B NM_006955 // NM_006955 0.260.10 0.018 ZNF33B // zinc finger protein 33B // 10q11.2 // 7582 ///ENST000003 3304475 ARL3 NM_004311 // NM_004311 0.26 0.08 0.008 ARL3 //ADP- ribosylation factor- like 3 // 10q23.3 // 403 /// ENST00 3364306SOX6 NM_017508 // NM_017508 0.26 0.08 0.010 SOX6 // SRY (sex determiningregion Y)-box 6 // 11p15.3 // 55553 // 3185498 SLC31A2 NM_001860 //NM_001860 0.25 0.09 0.015 SLC31A2 // solute carrier family 31 (coppertransporters), member 2 3998766 KAL1 NM_000216 // NM_000216 0.25 0.070.006 KAL1 // Kallmann syndrome 1 sequence // Xp22.32 // 3730 ///ENST000 3143266 PSKH2 NM_033126 // NM_033126 0.25 0.07 0.006 PSKH2 //protein serine kinase H2 // 8q21.2 // 85481 /// ENST000002 3458911CTDSP2 NM_005730 // NM_005730 0.25 0.06 0.003 CTDSP2 // CTD(carboxy-terminal domain, RNA polymerase II, polypept 3195034 PTGDSNM_000954 // NM_000954 0.25 0.08 0.010 PTGDS // prostaglandin D2synthase 21 kDa (brain) // 9q34.2-q34.3 // 3854066 C19orf42 NM_024104 //NM_024104 0.25 0.08 0.010 C19orf42 // chromosome 19 open reading frame42 // 19p13.11 // 7908 3819474 ANGPTL4 NM_139314 // NM_139314 0.25 0.060.004 ANGPTL4 // angiopoietin-like 4 // 19p13.3 // 51129 ///NM_001039667 3944084 TOM1 NM_005488 // NM_005488 0.25 0.07 0.006 TOM1 //target of myb1 (chicken) // 22q13.1 // 10043 /// ENST000003 3848243 INSRNM_000208 // NM_000208 0.24 0.09 0.014 INSR // insulin receptor //19p13.3-p13.2 // 3643 /// NM_001079817 3168415 CLTA NM_007096 //NM_007096 0.24 0.08 0.009 CLTA // clathrin, light chain (Lca) // 9p13 //1211 /// NM_00107667 2609462 CAV3 NM_033337 // NM_033337 0.24 0.07 0.007CAV3 // caveolin 3 // 3p25 // 859 /// NM_001234 // CAV3 // caveolin3393834 C11orf60 BC022856 // BC022856 0.24 0.06 0.003 C11orf60 //chromosome 11 open reading frame 60 // 11q23.3 // 56912 3755614 STAC2NM_198993 // NM_198993 0.24 0.07 0.009 STAC2 // SH3 and cysteine richdomain 2 // 17q12 // 342667 /// ENST 3627363 NARG2 NM_024611 //NM_024611 0.24 0.06 0.003 NARG2 // NMDA receptor regulated 2 // 15q22.2// 79664 /// NM_00101 3212976 ZCCHC6 NM_024617 // NM_024617 0.24 0.080.014 ZCCHC6 // zinc finger, CCHC domain containing 6 // 9q21 // 79670// 3275922 PRKCQ NM_006257 // NM_006257 0.24 0.05 0.002 PRKCQ // proteinkinase C, theta // 10p15 // 5588 /// ENST000002631 3023825 C7orf45BC017587 // BC017587 0.23 0.09 0.020 C7orf45 // chromosome 7 openreading frame 45 // 7q32.2 // 136263 // 3832906 IL29 NM_172140 //NM_172140 0.23 0.08 0.015 IL29 // interleukin 29 (interferon, lambda 1)// 19q13.13 // 282618 3529156 NGDN NM_015514 // NM_015514 0.23 0.080.012 NGDN // neuroguidin, EIF4E binding protein // 14q11.2 // 25983 ///2620448 CLEC3B NM_003278 // NM_003278 0.23 0.08 0.014 CLEC3B // C-typelectin domain family 3, member B // 3p22-p21.3 // 3481296 SGCG NM_000231// NM_000231 0.23 0.09 0.019 SGCG // sarcoglycan, gamma (35 kDadystrophin- associated glycoprotei 3135184 RB1CC1 NM_014781 // NM_0147810.23 0.07 0.008 RB1CC1 // RB1- inducible coiled- coil 1 // 8q11 // 9821/// NM_001083 2421843 GBP3 NM_018284 // NM_018284 0.23 0.06 0.004 GBP3// guanylate binding protein 3 // 1p22.2 // 2635 /// ENST00000 3385003CREBZF NM_001039618 // NM_001039618 0.23 0.09 0.020 CREBZF // CREB/ATFbZIP transcription factor // 11q14 // 58487 / 3610804 IGF1R NM_000875 //NM_000875 0.23 0.08 0.013 IGF1R // insulin- like growth factor 1receptor // 15q26.3 // 3480 / 3606304 AKAP13 NM_006738 // NM_006738 0.230.04 0.000 AKAP13 // A kinase (PRKA) anchor protein 13 // 15q24-q25 //11214 / 2565579 ANKRD39 NM_016466 // NM_016466 0.23 0.05 0.003 ANKRD39// ankyrin repeat domain 39 // 2q11.2 // 51239 /// ENST0000 2722151 RBPJNM_005349 // NM_005349 0.22 0.07 0.008 RBPJ // recombination signalbinding protein for immunoglobulin kap 3031533 GIMAP4 NM_018326 //NM_018326 0.22 0.08 0.017 GIMAP4 // GTPase, IMAP family member 4 //7q36.1 // 55303 /// ENST0 3725481 UBE2Z NM_023079 // NM_023079 0.22 0.060.004 UBE2Z // ubiquitin- conjugating enzyme E2Z // 17q21.32 // 65264/// 3549575 IFI27 NM_005532 // NM_005532 0.22 0.08 0.016 IFI27 //interferon, alpha-inducible protein 27 // 14q32 // 3429 // 3725035NFE2L1 NM_003204 // NM_003204 0.22 0.07 0.011 NFE2L1 // nuclear factor(erythroid- derived 2)-like 1 // 17q21.3 // 3348748 C11orf1 NM_022761 //NM_022761 0.22 0.07 0.008 C11orf1 // chromosome 11 open reading frame 1// 11q13-q22 // 64776 3722039 RAMP2 NM_005854 // NM_005854 0.22 0.050.003 RAMP2 // receptor (G protein-coupled) activity modifying protein 23886704 STK4 NM_006282 // NM_006282 0.22 0.07 0.012 STK4 //serine/threonine kinase 4 // 20q11.2-q13.2 // 6789 /// ENST 3645901FLJ14154 NM_024845 // NM_024845 0.22 0.06 0.005 FLJ14154 // hypotheticalprotein FLJ14154 // 16p13.3 // 79903 /// N 3367673 MPPED2 NM_001584 //NM_001584 0.22 0.08 0.017 MPPED2 // metallophosphoesterase domaincontaining 2 // 11p13 // 74 3219885 PTPN3 NM_002829 // NM_002829 0.220.05 0.003 PTPN3 // protein tyrosine phosphatase, non- receptor type 3// 9q31 3791466 — — 0.22 0.06 0.007 3717635 ZNF207 NM_001098507 //NM_001098507 0.22 0.08 0.015 ZNF207 // zinc finger protein 207 //17q11.2 // 7756 /// NM_0034 2648141 MBNL1 NM_021038 // NM_021038 0.220.07 0.009 MBNL1 // muscleblind-like (Drosophila) // 3q25 // 4154 ///NM_20729 2436938 PBXIP1 NM_020524 // NM_020524 0.21 0.05 0.002 PBXIP1 //pre-B- cell leukemia homeobox interacting protein 1 // 1q2 3299705 PANK1NM_148977 // NM_148977 0.21 0.06 0.007 PANK1 // pantothenate kinase 1 //10q23.31 // 53354 /// NM_148978 / 3628923 FAM96A NM_032231 // NM_0322310.21 0.05 0.003 FAM96A // family with sequence similarity 96, member A// 15q22.31 2353669 CD2 NM_001767 // CD2 NM_001767 0.21 0.06 0.006 //CD2 molecule // 1p13 // 914 /// ENST00000369478 // CD2 // CD 3474450PLA2G1B NM_000928 // NM_000928 0.21 0.08 0.016 PLA2G1B // phospholipaseA2, group IB (pancreas) // 12q23-q24.1 // 3722417 NBR1 NM_031858 //NM_031858 0.21 0.08 0.017 NBR1 // neighbor of BRCA1 gene 1 // 17q21.31// 4077 /// NM_005899 3234760 CUGBP2 NM_001025077 // NM_001025077 0.210.06 0.004 CUGBP2 // CUG triplet repeat, RNA binding protein 2 // 10p13// 3627422 RORA NM_134260 // NM_134260 0.21 0.06 0.006 RORA // RAR-related orphan receptor A // 15q21-q22 // 6095 /// NM_0 3382061 XRRA1NM_182969 // NM_182969 0.21 0.08 0.017 XRRA1 // X-ray radiationresistance associated 1 // 11q13.4 // 1435 3015338 STAG3 NM_012447 //NM_012447 0.21 0.06 0.007 STAG3 // stromal antigen 3 // 7q22.1 // 10734/// ENST00000317296 / 2665720 ZNF385D NM_024697 // NM_024697 0.21 0.070.013 ZNF385D // zinc finger protein 385D // 3p24.3 // 79750 ///ENST0000 3154185 TMEM71 NM_144649 // NM_144649 0.21 0.06 0.009 TMEM71 //transmembrane protein 71 // 8q24.22 // 137835 /// ENST000 3789947 NEDD4LNM_015277 // NM_015277 0.21 0.08 0.016 NEDD4L // neural precursor cellexpressed, developmentally down-reg 2688933 CD200R2 ENST00000383679ENST00000383679 0.21 0.08 0.016 // CD200R2 // CD200 cell surfaceglycoprotein receptor isoform 2 3379644 CPT1A NM_001876 // NM_0018760.21 0.04 0.001 CPT1A // carnitine palmitoyltransferase 1A (liver) //11q13.1-q13.2 3677795 CREBBP NM_004380 // NM_004380 0.21 0.05 0.004CREBBP // CREB binding protein (Rubinstein-Taybi syndrome) // 16p132358320 TARS2 NM_025150 // NM_025150 0.21 0.06 0.007 TARS2 // threonyl-tRNA synthetase 2, mitochondrial (putative) // 1q 3228373 TSC1 NM_000368// NM_000368 0.20 0.06 0.006 TSC1 // tuberous sclerosis 1 // 9q34 //7248 /// NM_001008567 // TS 3362795 RNF141 NM_016422 // NM_016422 0.200.08 0.019 RNF141 // ring finger protein 141 // 11p15.4 // 50862 ///ENST00000 3673684 CDT1 NM_030928 // NM_030928 0.20 0.07 0.015 CDT1 //chromatin licensing and DNA replication factor 1 // 16q24.3 3042881HOXA7 NM_006896 // NM_006896 0.20 0.02 0.000 HOXA7 // homeobox A7 //7p15-p14 // 3204 /// ENST00000396347 // HOX 3381817 UCP2 NM_003355 //NM_003355 0.20 0.05 0.005 UCP2 // uncoupling protein 2 (mitochondrial,proton carrier) // 11q 3415068 ANKRD33 NM_182608 // NM_182608 0.20 0.060.006 ANKRD33 // ankyrin repeat domain 33 // 12q13.13 // 341405 ///ENST0 3633403 SIN3A NM_015477 // NM_015477 0.20 0.07 0.014 SIN3A // SIN3homolog A, transcription regulator (yeast) // 15q24.2 3380901 NUMA1NM_006185 // NM_006185 0.19 0.04 0.002 NUMA1 // nuclear mitoticapparatus protein 1 // 11q13 // 4926 /// E 2598099 BARD1 NM_000465 //NM_000465 0.19 0.07 0.015 BARD1 // BRCA1 associated RING domain 1 //2q34-q35 // 580 /// ENST 3139722 NCOA2 NM_006540 // NM_006540 0.19 0.060.010 NCOA2 // nuclear receptor coactivator 2 // 8q13.3 // 10499 ///ENST 3641871 LINS1 NM_018148 // NM_018148 0.19 0.06 0.013 LINS1 // lineshomolog 1 (Drosophila) // 15q26.3 // 55180 /// NM_00 3401217 TULP3NM_003324 // NM_003324 0.19 0.06 0.008 TULP3 // tubby like protein 3 //12p13.3 // 7289 /// ENST0000022824 3741997 ANKFY1 NM_016376 // NM_0163760.19 0.06 0.008 ANKFY1 // ankyrin repeat and FYVE domain containing 1 //17p13.3 // 2622742 C3orf45 BC028000 // BC028000 0.19 0.06 0.013 C3orf45// chromosome 3 open reading frame 45 // 3p21.31 // 132228 / 3845352UQCR NM_006830 // NM_006830 0.19 0.06 0.014 UQCR // ubiquinol-cytochrome c reductase, 6.4 kDa subunit // 19p13.3 3960356 BAIAP2L2NM_025045 // NM_025045 0.19 0.07 0.018 BAIAP2L2 // BAI1-associatedprotein 2-like 2 // 22q13.1 // 80115 // 3645947 CLUAP1 NM_015041 //NM_015041 0.19 0.06 0.012 CLUAP1 // clusterin associated protein 1 //16p13.3 // 23059 /// NM 3835544 ZNF227 NM_182490 // NM_182490 0.18 0.060.011 ZNF227 // zinc finger protein 227 // — // 7770 /// ENST00000313043368748 FBXO3 NM_033406 // NM_033406 0.18 0.07 0.020 FBXO3 // F-boxprotein 3 // 11p13 // 26273 /// NM_012175 // FBXO3 / 3621623 ELL3NM_025165 // NM_025165 0.18 0.05 0.005 ELL3 // elongation factor RNApolymerase II-like 3 // 15q15.3 // 80 3430552 PWP1 NM_007062 //NM_007062 0.18 0.07 0.016 PWP1 // PWP1 homolog (S. cerevisiae) //12q23.3 // 11137 /// ENST00 2844908 BTNL9 NM_152547 // NM_152547 0.180.05 0.005 BTNL9 // butyrophilin-like 9 // 5q35.3 // 153579 ///ENST0000032770 4021508 ZNF280C NM_017666 // NM_017666 0.18 0.07 0.018ZNF280C // zinc finger protein 280C // Xq25 // 55609 /// ENST0000032489071 TET3 NM_144993 // NM_144993 0.18 0.04 0.003 TET3 // tet oncogenefamily member 3 // 2p13.1 // 200424 /// ENST00 2516879 HOXD8 NM_019558// NM_019558 0.18 0.06 0.015 HOXD8 // homeobox D8 // 2q31.1 // 3234 ///ENST00000313173 // HOXD8 3740704 SMYD4 NM_052928 // NM_052928 0.18 0.060.012 SMYD4 // SET and MYND domain containing 4 // 17p13.3 // 114826 ///3975467 UTX NM_021140 // NM_021140 0.18 0.06 0.013 UTX // ubiquitouslytranscribed tetratricopeptide repeat, X chromos 3699044 RFWD3 NM_018124// NM_018124 0.18 0.06 0.011 RFWD3 // ring finger and WD repeat domain 3// 16q22.3 // 55159 /// 3473083 MED13L NM_015335 // NM_015335 0.18 0.020.000 MED13L // mediator complex subunit 13-like // 12q24.21 // 23389/// 2332711 PPIH NM_006347 // NM_006347 0.17 0.06 0.017 PPIH //peptidylprolyl isomerase H (cyclophilin H) // 1p34.1 // 104 3556990 JUBNM_032876 // JUB NM_032876 0.17 0.04 0.004 // jub, ajuba homolog(Xenopus laevis) // 14q11.2 // 84962 /// 2780143 BDH2 NM_020139 //NM_020139 0.17 0.05 0.006 BDH2 // 3- hydroxybutyrate dehydrogenase, type2 // 4q24 // 56898 // 3899495 C20orf12 NM_001099407 // NM_001099407 0.170.05 0.008 C20orf12 // chromosome 20 open reading frame 12 // 20p11.23// 5 3290875 ANK3 NM_020987 // NM_020987 0.17 0.03 0.001 ANK3 // ankyrin3, node of Ranvier (ankyrin G) // 10q21 // 288 /// 3576014 C14orf102NM_017970 // NM_017970 0.17 0.04 0.002 C14orf102 // chromosome 14 openreading frame 102 // 14q32.11 // 55 3644887 ATP6V0C NM_001694 //NM_001694 0.17 0.06 0.017 ATP6V0C // ATPase, H+ transporting, lysosomal16 kDa, V0 subunit c / 2648378 RAP2B NM_002886 // NM_002886 0.17 0.060.017 RAP2B // RAP2B, member of RAS oncogene family // 3q25.2 // 5912/// 2362892 ATP1A2 NM_000702 // NM_000702 0.16 0.06 0.015 ATP1A2 //ATPase, Na+/K+ transporting, alpha 2 (+) polypeptide // 1 2361488 RHBGNM_020407 // NM_020407 0.16 0.06 0.014 RHBG // Rh family, B glycoprotein// 1q21.3 // 57127 /// ENST000003 3415915 PFDN5 NM_002624 // NM_0026240.16 0.05 0.011 PFDN5 // prefoldin subunit 5 // 12q12 // 5204 ///NM_145897 // PFDN 3433796 PEBP1 NM_002567 // NM_002567 0.16 0.04 0.004PEBP1 // phosphatidylethanolamine binding protein 1 // 12q24.23 //3788302 SMAD4 NM_005359 // NM_005359 0.16 0.05 0.012 SMAD4 // SMADfamily member 4 // 18q21.1 // 4089 /// ENST0000039841 3436236 ZNF664NM_152437 // NM_152437 0.16 0.06 0.016 ZNF664 // zinc finger protein 664// 12q24.31 // 144348 /// ENST000 3441542 TMEM16B NM_020373 // NM_0203730.16 0.06 0.018 TMEM16B // transmembrane protein 16B // 12p13.3 // 57101/// ENST00 3456353 CALCOCO1 NM_020898 // NM_020898 0.16 0.05 0.010CALCOCO1 // calcium binding and coiled-coil domain 1 // 12q13.13 //3888721 PTPN1 NM_002827 // NM_002827 0.16 0.06 0.020 PTPN1 // proteintyrosine phosphatase, non- receptor type 1 // 20q13 3138204 CYP7B1NM_004820 // NM_004820 0.15 0.05 0.014 CYP7B1 // cytochrome P450, family7, subfamily B, polypeptide 1 // 3278401 FRMD4A NM_018027 // NM_0180270.15 0.05 0.009 FRMD4A // FERM domain containing 4A // 10p13 // 55691/// ENST00000 3904226 RBM39 NM_184234 // NM_184234 0.15 0.05 0.015 RBM39// RNA binding motif protein 39 // 20q11.22 // 9584 /// NM_00 3791850SERPINB13 NM_012397 // NM_012397 0.15 0.04 0.005 SERPINB13 // serpinpeptidase inhibitor, clade B (ovalbumin), membe 3665603 CTCF NM_006565// NM_006565 0.15 0.04 0.004 CTCF // CCCTC- binding factor (zinc fingerprotein) // 16q21-q22.3 / 3969802 BMX NM_203281 // NM_203281 0.15 0.050.016 BMX // BMX non- receptor tyrosine kinase // Xp22.2 // 660 ///NM_001 3621276 HISPPD2A NM_014659 // NM_014659 0.14 0.04 0.005 HISPPD2A// histidine acid phosphatase domain containing 2A // 15q1 2325113C1orf213 NM_138479 // NM_138479 0.14 0.05 0.012 C1orf213 // chromosome 1open reading frame 213 // 1p36.12 // 14889 3681956 KIAA0430 NM_014647 //NM_014647 0.14 0.05 0.018 KIAA0430 // KIAA0430 // 16p13.11 // 9665 ///ENST00000396368 // KIA 3415193 GRASP NM_181711 // NM_181711 0.14 0.050.019 GRASP // GRP1 (general receptor for phosphoinositides1)-associated 3249369 LRRTM3 NM_178011 // NM_178011 0.14 0.05 0.011LRRTM3 // leucine rich repeat transmembrane neuronal 3 // 10q21.3 /3874023 PTPRA NM_002836 // NM_002836 0.14 0.04 0.004 PTPRA // proteintyrosine phosphatase, receptor type, A // 20p13 // 3809621 FECHNM_001012515 // NM_001012515 0.14 0.04 0.009 FECH // ferrochelatase(protoporphyria) // 18q21.3 // 2235 /// N 3351385 MLL NM_005933 //NM_005933 0.14 0.05 0.016 MLL // myeloid/lymphoid or mixed-lineageleukemia (trithorax homolo 3288707 ERCC6 NM_000124 // NM_000124 0.140.05 0.016 ERCC6 // excision repair cross- complementing rodent repairdeficien 3624607 MYO5A NM_000259 // NM_000259 0.14 0.04 0.006 MYO5A //myosin VA (heavy chain 12, myoxin) // 15q21 // 4644 /// EN 3353859 OR4D5NM_001001965 // NM_001001965 0.14 0.05 0.017 OR4D5 // olfactoryreceptor, family 4, subfamily D, member 5 // 2823797 TSLP NM_033035 //NM_033035 0.14 0.05 0.013 TSLP // thymic stromal lymphopoietin // 5q22.1// 85480 /// NM_1385 2414366 PPAP2B NM_003713 // NM_003713 0.13 0.040.007 PPAP2B // phosphatidic acid phosphatase type 2B // 1pter-p22.1 //8 3878308 CSRP2BP NM_020536 // NM_020536 0.13 0.05 0.019 CSRP2BP //CSRP2 binding protein // 20p11.23 // 57325 /// NM_177926 4025771 CD99L2NM_031462 // NM_031462 0.13 0.04 0.007 CD99L2 // CD99 molecule-like 2 //Xq28 // 83692 /// NM_134446 // CD 3414776 LETMD1 NM_015416 // NM_0154160.13 0.05 0.014 LETMD1 // LETM1 domain containing 1 // 12q13.13 // 25875/// NM_001 3645253 SRRM2 NM_016333 // NM_016333 0.13 0.04 0.007 SRRM2 //serine/arginine repetitive matrix 2 // 16p13.3 // 23524 // 2440700ADAMTS4 NM_005099 // NM_005099 0.13 0.03 0.005 ADAMTS4 // ADAMmetallopeptidase with thrombospondin type 1 motif, 2609870 BRPF1NM_001003694 // NM_001003694 0.13 0.04 0.012 BRPF1 // bromodomain andPHD finger containing, 1 // 3p26-p25 // 3632298 ADPGK NM_031284 //NM_031284 0.13 0.04 0.007 ADPGK // ADP- dependent glucokinase // 15q24.1// 83440 /// ENST0000 3184940 GNG10 NM_001017998 // NM_001017998 0.130.04 0.011 GNG10 // guanine nucleotide binding protein (G protein),gamma 1 3223776 C5 NM_001735 // C5 NM_001735 0.13 0.04 0.008 //complement component 5 // 9q33-q34 // 727 /// ENST00000223642 3922100MX1 NM_002462 // NM_002462 0.12 0.04 0.015 MX1 // myxovirus (influenzavirus) resistance 1, interferon-inducib 3960478 CSNK1E NM_001894 //NM_001894 0.12 0.04 0.018 CSNK1E // casein kinase 1, epsilon // 22q13.1// 1454 /// NM_152221 3715703 SUPT6H NM_003170 // NM_003170 0.11 0.030.005 SUPT6H // suppressor of Ty 6 homolog (S. cerevisiae) // 17q11.2 //2322818 PADI3 NM_016233 // NM_016233 0.11 0.03 0.006 PADI3 // peptidylarginine deiminase, type III // 1p36.13 // 51702 2393740 KIAA0562NM_014704 // NM_014704 0.11 0.03 0.009 KIAA0562 // KIAA0562 // 1p36.32// 9731 /// ENST00000378230 // KIAA 3784509 ZNF271 NM_001112663 //NM_001112663 0.11 0.04 0.020 ZNF271 // zinc finger protein 271 // 18q12// 10778 /// NM_00662 3372253 CUGBP1 NM_006560 // NM_006560 0.11 0.040.011 CUGBP1 // CUG triplet repeat, RNA binding protein 1 // 11p11 //106 2948259 TRIM26 NM_003449 // NM_003449 0.11 0.03 0.006 TRIM26 //tripartite motif-containing 26 // 6p21.3 // 7726 /// ENST 3191900 NUP214NM_005085 // NM_005085 0.11 0.03 0.003 NUP214 // nucleoporin 214 kDa //9q34.1 // 8021 /// ENST00000359428 3105581 CA3 NM_005181 // CA3NM_005181 0.11 0.03 0.003 // carbonic anhydrase III, muscle specific //8q13-q22 // 761 / 3832457 RYR1 NM_000540 // NM_000540 0.11 0.03 0.006RYR1 // ryanodine receptor 1 (skeletal) // 19q13.1 // 6261 /// NM_03936256 BCL2L13 NM_015367 // NM_015367 0.10 0.02 0.002 BCL2L13 // BCL2-like 13 (apoptosis facilitator) // 22q11 // 23786 / 3599280 PIAS1NM_016166 // NM_016166 0.10 0.04 0.017 PIAS1 // protein inhibitor ofactivated STAT, 1 // 15q // 8554 /// 3755976 MED24 NM_014815 //NM_014815 0.10 0.04 0.019 MED24 // mediator complex subunit 24 //17q21.1 // 9862 /// NM_0010 3656418 SRCAP NM_006662 // NM_006662 0.100.04 0.017 SRCAP // Snf2- related CREBBP activator protein // 16p11.2 //10847 3943101 DEPDC5 NM_014662 // NM_014662 0.09 0.01 0.000 DEPDC5 //DEP domain containing 5 // 22q12.3 // 9681 /// NM_0010071 3960685 DMC1NM_007068 // NM_007068 0.09 0.03 0.013 DMC1 // DMC1 dosage suppressor ofmck1 homolog, meiosis-specific ho 2434776 CDC42SE1 NM_001038707 //NM_001038707 0.08 0.03 0.014 CDC42SE1 // CDC42 small effector 1 //1q21.2 // 56882 /// NM_020 3438417 SFRS8 NM_004592 // NM_004592 0.080.03 0.016 SFRS8 // splicing factor, arginine/serine-rich 8(suppressor-of- whi 3457696 PAN2 NM_014871 // NM_014871 0.08 0.02 0.008PAN2 // PAN2 polyA specific ribonuclease subunit homolog (S. cerevi2534615 SCLY NM_016510 // NM_016510 0.08 0.02 0.004 SCLY //selenocysteine lyase // 2q37.3 // 51540 /// ENST00000254663 2765865RELL1 NM_001085400 // NM_001085400 0.07 0.02 0.002 RELL1 // RELT- like 1// 4p14 // 768211 /// NM_001085399 // RELL1 3765642 INTS2 NM_020748 //NM_020748 0.05 0.01 0.005 INTS2 // integrator complex subunit 2 //17q23.2 // 57508 /// ENST0 2906607 NFYA NM_002505 // NM_002505 −0.070.02 0.011 NFYA // nuclear transcription factor Y, alpha // 6p21.3 //4800 /// 3168102 CREB3 NM_006368 // NM_006368 −0.07 0.02 0.010 CREB3 //cAMP responsive element binding protein 3 // 9pter-p22.1 / 3939365SMARCB1 NM_003073 // NM_003073 −0.07 0.02 0.013 SMARCB1 // SWI/SNFrelated, matrix associated, actin dependent regu 3415229 NR4A1 NM_002135// NM_002135 −0.07 0.03 0.015 NR4A1 // nuclear receptor subfamily 4,group A, member 1 // 12q13 / 2437801 ARHGEF2 NM_004723 // NM_004723−0.09 0.02 0.002 ARHGEF2 // rho/rac guanine nucleotide exchange factor(GEF) 2 // 1q 3645565 THOC6 NM_024339 // NM_024339 −0.10 0.04 0.018THOC6 // THO complex 6 homolog (Drosophila) // 16p13.3 // 79228 ///2406766 MRPS15 NM_031280 // NM_031280 −0.11 0.03 0.003 MRPS15 //mitochondrial ribosomal protein S15 // 1p35-p34.1 // 6496 3553141KIAA0329 NM_014844 // NM_014844 −0.11 0.04 0.018 KIAA0329 // KIAA0329 //14q32.31 // 9895 /// ENST00000359520 // KIA 3297666 DYDC1 NM_138812 //NM_138812 −0.11 0.02 0.000 DYDC1 // DPY30 domain containing 1 // 10q23.1// 143241 /// ENST000 3625674 RFXDC2 NM_022841 // NM_022841 −0.12 0.040.012 RFXDC2 // regulatory factor X domain containing 2 // 15q21.3 //648 2926969 PDE7B NM_018945 // NM_018945 −0.12 0.04 0.013 PDE7B //phosphodiesterase 7B // 6q23-q24 // 27115 /// ENST00000308 3525313COL4A1 NM_001845 // NM_001845 −0.12 0.04 0.014 COL4A1 // collagen, typeIV, alpha 1 // 13q34 // 1282 /// ENST00000 2438892 FCRL5 NM_031281 //NM_031281 −0.12 0.04 0.009 FCRL5 // Fc receptor-like 5 // 1q21 // 83416/// ENST00000361835 // 3220846 SUSD1 NM_022486 // NM_022486 −0.12 0.030.006 SUSD1 // sushi domain containing 1 // 9q31.3-q33.1 // 64420 ///ENS 3598430 SLC24A1 NM_004727 // NM_004727 −0.12 0.05 0.019 SLC24A1 //solute carrier family 24 (sodium/potassium/ calcium excha 3506431 RNF6NM_005977 // NM_005977 −0.12 0.04 0.011 RNF6 // ring finger protein(C3H2C3 type) 6 // 13q12.2 // 6049 /// 3696057 SLC12A4 NM_005072 //NM_005072 −0.12 0.02 0.001 SLC12A4 // solute carrier family 12(potassium/chloride transporter 2519577 COL3A1 NM_000090 // NM_000090−0.12 0.04 0.012 COL3A1 // collagen, type III, alpha 1 (Ehlers- Danlossyndrome type 3734479 TMEM104 NM_017728 // NM_017728 −0.13 0.04 0.015TMEM104 // transmembrane protein 104 // 17q25.1 // 54868 /// ENST003345157 PIWIL4 NM_152431 // NM_152431 −0.13 0.05 0.015 PIWIL4 //piwi-like 4 (Drosophila) // 11q21 // 143689 /// ENST00000 2949471 NEU1NM_000434 // NM_000434 −0.13 0.04 0.013 NEU1 // sialidase 1 (lysosomalsialidase) // 6p21.3 // 4758 /// ENS 2599670 CRYBA2 NM_057093 //NM_057093 −0.13 0.04 0.014 CRYBA2 // crystallin, beta A2 // 2q34-q36 //1412 /// NM_005209 // 3922444 ABCG1 NM_207628 // NM_207628 −0.13 0.030.003 ABCG1 // ATP- binding cassette, sub-family G (WHITE), member 1 //21 2760371 WDR1 NM_017491 // NM_017491 −0.14 0.05 0.019 WDR1 // WDrepeat domain 1 // 4p16.1 // 9948 /// NM_005112 // WDR1 2835440 TCOF1NM_001008656 // NM_001008656 −0.14 0.04 0.007 TCOF1 // Treacher Collins-Franceschetti syndrome 1 // 5q32-q33.1 2451544 MYOG NM_002479 //NM_002479 −0.14 0.05 0.018 MYOG // myogenin (myogenic factor 4) //1q31-q41 // 4656 /// ENST00 3745504 SCO1 NM_004589 // NM_004589 −0.140.03 0.003 SCO1 // SCO cytochrome oxidase deficient homolog 1 (yeast) //17p12 2835213 PPARGC1B NM_133263 // NM_133263 −0.14 0.04 0.006 PPARGC1B// peroxisome proliferator- activated receptor gamma, coact 3704567CBFA2T3 NM_005187 // NM_005187 −0.14 0.05 0.020 CBFA2T3 // core- bindingfactor, runt domain, alpha subunit 2; trans 2893562 RREB1 NM_002955 //NM_002955 −0.14 0.04 0.006 RREB1 // ras responsive element bindingprotein 1 // 6p25 // 6239 / 2672712 SCAP NM_012235 // NM_012235 −0.140.04 0.009 SCAP // SREBF chaperone // 3p21.31 // 22937 ///ENST00000265565 // 2768197 CORIN NM_006587 // NM_006587 −0.14 0.05 0.011CORIN // corin, serine peptidase // 4p13-p12 // 10699 /// ENST000002495279 VWA3B NM_144992 // NM_144992 −0.14 0.04 0.006 VWA3B // vonWillebrand factor A domain containing 3B // 2q11.2 // 2903588 PFDN6NM_014260 // NM_014260 −0.14 0.05 0.014 PFDN6 // prefoldin subunit 6 //6p21.3 // 10471 /// ENST00000399112 3031383 REPIN1 NM_013400 //NM_013400 −0.15 0.05 0.018 REPIN1 // replication initiator 1 // 7q36.1// 29803 /// NM_014374 3754469 ACACA NM_198839 // NM_198839 −0.15 0.050.010 ACACA // acetyl- Coenzyme A carboxylase alpha // 17q21 // 31 ///NM_(—) 3767480 AXIN2 NM_004655 // NM_004655 −0.15 0.05 0.013 AXIN2 //axin 2 (conductin, axil) // 17q23-q24 // 8313 /// ENST0000 2954506 CRIP3NM_206922 // NM_206922 −0.15 0.06 0.018 CRIP3 // cysteine- rich protein3 // 6p21.1 // 401262 /// ENST000003 3845263 ADAMTSL5 NM_213604 //NM_213604 −0.15 0.06 0.016 ADAMTSL5 // ADAMTS-like 5 // 19p13.3 //339366 /// ENST00000330475 2565143 STARD7 NM_020151 // NM_020151 −0.150.06 0.016 STARD7 // StAR- related lipid transfer (START) domaincontaining 7 / 2321960 PLEKHM2 NM_015164 // NM_015164 −0.16 0.05 0.009PLEKHM2 // pleckstrin homology domain containing, family M (with RU3829174 GPATCH1 NM_018025 // NM_018025 −0.16 0.03 0.001 GPATCH1 // Gpatch domain containing 1 // 19q13.11 // 55094 /// ENS 2798586 AHRRNM_020731 // NM_020731 −0.16 0.05 0.011 AHRR // aryl- hydrocarbonreceptor repressor // 5p15.3 // 57491 /// 2362991 CASQ1 NM_001231 //NM_001231 −0.16 0.06 0.015 CASQ1 // calsequestrin 1 (fast-twitch,skeletal muscle) // 1q21 // 3954525 ZNF280B NM_080764 // NM_080764 −0.160.04 0.005 ZNF280B // zinc finger protein 280B // 22q11.22 // 140883 ///ENST0 4020991 ACTRT1 NM_138289 // NM_138289 −0.16 0.05 0.007 ACTRT1 //actin- related protein T1 // Xq25 // 139741 /// ENST000003 3982975POU3F4 NM_000307 // NM_000307 −0.16 0.05 0.013 POU3F4 // POU class 3homeobox 4 // Xq21.1 // 5456 /// ENST00000373 3963990 PKDREJ NM_006071// NM_006071 −0.16 0.03 0.001 PKDREJ // polycystic kidney disease(polycystin) and REJ homolog (s 2436401 JTB NM_006694 // JTB NM_006694−0.16 0.06 0.014 // jumping translocation breakpoint // 1q21 // 10899/// NM_002 2759654 ABLIM2 NM_032432 // NM_032432 −0.16 0.05 0.007 ABLIM2// actin binding LIM protein family, member 2 // 4p16-p15 // 2437329CLK2 NM_003993 // NM_003993 −0.16 0.06 0.016 CLK2 // CDC-like kinase 2// 1q21 // 1196 /// NR_002711 // CLK2P // 3401119 ITFG2 NM_018463 //NM_018463 −0.16 0.04 0.004 ITFG2 // integrin alpha FG-GAP repeatcontaining 2 // 12p13.33 // 5 3599709 GLCE NM_015554 // NM_015554 −0.160.06 0.014 GLCE // glucuronic acid epimerase // 15q23 // 26035 ///ENST0000026 3882413 C20orf114 NM_033197 // NM_033197 −0.16 0.06 0.020C20orf114 // chromosome 20 open reading frame 114 // 20q11.21 // 923712922 C17orf39 NM_024052 // NM_024052 −0.16 0.06 0.017 C17orf39 //chromosome 17 open reading frame 39 // 17p11.2 // 79018 2473376 EFR3BBC049384 // BC049384 −0.17 0.05 0.009 EFR3B // EFR3 homolog B (S.cerevisiae) // 2p23.3 // 22979 /// ENST0 2607262 STK25 NM_006374 //NM_006374 −0.17 0.06 0.015 STK25 // serine/threonine kinase 25 (STE20homolog, yeast) // 2q37. 3755580 CACNB1 NM_199247 // NM_199247 −0.170.06 0.013 CACNB1 // calcium channel, voltage-dependent, beta 1 subunit// 17q 3402150 NTF3 NM_001102654 // NM_001102654 −0.17 0.06 0.020 NTF3// neurotrophin 3 // 12p13 // 4908 /// NM_002527 // NTF3 // 3014714ARPC1B NM_005720 // NM_005720 −0.17 0.06 0.020 ARPC1B // actin relatedprotein 2/3 complex, subunit 1B, 41 kDa // 7 3723071 DBF4B NM_145663 //NM_145663 −0.17 0.04 0.002 DBF4B // DBF4 homolog B (S. cerevisiae) //17q21.31|17q21 // 80174 2371255 SMG7 NM_173156 // NM_173156 −0.17 0.060.014 SMG7 // Smg-7 homolog, nonsense mediated mRNA decay factor (C.eleg 3217487 ALG2 NM_033087 // NM_033087 −0.17 0.06 0.011 ALG2 //asparagine-linked glycosylation 2 homolog (S. cerevisiae, a 3352159LOC100130353 AK130019 // AK130019 −0.17 0.06 0.018 LOC100130353 //hypothetical protein LOC100130353 // 11q23.3 // 1001 3401259 TEAD4NM_003213 // NM_003213 −0.17 0.07 0.020 TEAD4 // TEA domain familymember 4 // 12p13.3-p13.2 // 7004 /// NM 3114618 RNF139 NM_007218 //NM_007218 −0.17 0.06 0.015 RNF139 // ring finger protein 139 // 8q24 //11236 /// ENST00000303 2991150 TSPAN13 NM_014399 // NM_014399 −0.18 0.050.006 TSPAN13 // tetraspanin 13 // 7p21.1 // 27075 /// ENST00000262067// 2875193 P4HA2 NM_004199 // NM_004199 −0.18 0.05 0.007 P4HA2 //procollagen- proline, 2- oxoglutarate 4- dioxygenase (proline 4011743SLC7A3 NM_032803 // NM_032803 −0.18 0.06 0.009 SLC7A3 // solute carrierfamily 7 (cationic amino acid transporter, 3194015 LCN9 NM_001001676 //NM_001001676 −0.18 0.06 0.011 LCN9 // lipocalin 9 // 9q34.3 // 392399/// ENST00000277526 // L 3741040 MNT NM_020310 // NM_020310 −0.18 0.040.003 MNT // MAX binding protein // 17p13.3 // 4335 /// ENST00000174618/ 3901851 ABHD12 NM_001042472 // NM_001042472 −0.18 0.05 0.004 ABHD12 //abhydrolase domain containing 12 // 20p11.21 // 26090 2324919 EPHB2NM_017449 // NM_017449 −0.18 0.06 0.010 EPHB2 // EPH receptor B2 //1p36.1-p35 // 2048 /// NM_004442 // EPH 3185976 COL27A1 NM_032888 //NM_032888 −0.18 0.06 0.009 COL27A1 // collagen, type XXVII, alpha 1 //9q32 // 85301 /// ENST0 2855434 C5orf39 NM_001014279 // NM_001014279−0.18 0.05 0.007 C5orf39 // chromosome 5 open reading frame 39 // 5p12// 389289 2334476 MAST2 NM_015112 // NM_015112 −0.18 0.02 0.000 MAST2 //microtubule associated serine/threonine kinase 2 // 1p34.1 3962734 TTLL1NM_001008572 // NM_001008572 −0.18 0.03 0.001 TTLL1 // tubulin tyrosineligase-like family, member 1 // 22q13. 4017538 COL4A6 NM_033641 //NM_033641 −0.18 0.03 0.000 COL4A6 // collagen, type IV, alpha 6 // Xq22// 1288 /// NM_001847 3141589 IL7 NM_000880 // IL7 NM_000880 −0.19 0.050.006 // interleukin 7 // 8q12-q13 // 3574 /// ENST00000263851 // IL72436826 KCNN3 NM_002249 // NM_002249 −0.19 0.06 0.008 KCNN3 // potassiumintermediate/small conductance calcium-activated 3521174 ABCC4 NM_005845// NM_005845 −0.19 0.07 0.017 ABCC4 // ATP- binding cassette, sub-familyC (CFTR/MRP), member 4 // 3768280 C17orf58 NM_181656 // NM_181656 −0.190.07 0.017 C17orf58 // chromosome 17 open reading frame 58 // 17q24.2 //28401 2363784 HSPA6 NM_002155 // NM_002155 −0.19 0.06 0.011 HSPA6 //heat shock 70 kDa protein 6 (HSP70B′) // 1q23 // 3310 /// E 3928211GRIK1 NM_175611 // NM_175611 −0.19 0.06 0.011 GRIK1 // glutamatereceptor, ionotropic, kainate 1 // 21q22.11 // 2 2758978 EVC2 NM_147127// NM_147127 −0.19 0.06 0.012 EVC2 // Ellis van Creveld syndrome 2(limbin) // 4p16.2-p16.1 // 13 3740664 C17orf91 NM_032895 // NM_032895−0.19 0.07 0.015 C17orf91 // chromosome 17 open reading frame 91 //17p13.3 // 84981 2782267 NEUROG2 NM_024019 // NM_024019 −0.20 0.06 0.010NEUROG2 // neurogenin 2 // 4q25 // 63973 /// ENST00000313341 // NEU3826542 ZNF738 BC034499 // BC034499 −0.20 0.05 0.003 ZNF738 // zincfinger protein 738 // 19p12 // 148203 /// AK291002 // 3966000 TYMPNM_001113756 // NM_001113756 −0.20 0.05 0.003 TYMP // thymidinephosphorylase // 22q13|22q13.33 // 1890 /// NM 3607447 ABHD2 NM_007011// NM_007011 −0.20 0.05 0.005 ABHD2 // abhydrolase domain containing 2// 15q26.1 // 11057 /// NM 3236448 SUV39H2 NM_024670 // NM_024670 −0.200.07 0.011 SUV39H2 // suppressor of variegation 3-9 homolog 2(Drosophila) // 2528504 SPEG NM_005876 // NM_005876 −0.20 0.06 0.009SPEG // SPEG complex locus // 2q35 // 10290 /// ENST00000312358 //2730746 SLC4A4 NM_001098484 // NM_001098484 −0.20 0.06 0.007 SLC4A4 //solute carrier family 4, sodium bicarbonate cotranspor 2544662 DNMT3ANM_175629 // NM_175629 −0.20 0.06 0.007 DNMT3A // DNA (cytosine-5-)-methyltransferase 3 alpha // 2p23 // 17 2937625 C6orf208 BC101251 //BC101251 −0.20 0.06 0.007 C6orf208 // chromosome 6 open reading frame208 // 6q27 // 80069 /// 3233157 UCN3 NM_053049 // NM_053049 −0.20 0.080.017 UCN3 // urocortin 3 (stresscopin) // 10p15.1 // 114131 ///ENST0000 2548172 FEZ2 NM_001042548 // NM_001042548 −0.21 0.03 0.000 FEZ2// fasciculation and elongation protein zeta 2 (zygin II) / 3877809 OTORNM_020157 // NM_020157 −0.21 0.08 0.019 OTOR // otoraplin //20p12.1-p11.23 // 56914 /// ENST00000246081 // 3839400 C19orf63NM_175063 // NM_175063 −0.21 0.04 0.002 C19orf63 // chromosome 19 openreading frame 63 // 19q13.33 // 2843 3875108 C20orf196 AK292708 //AK292708 −0.21 0.06 0.006 C20orf196 // chromosome 20 open reading frame196 // 20p12.3 // 1498 2970985 TSPYL4 NM_021648 // NM_021648 −0.21 0.070.011 TSPYL4 // TSPY- like 4 // 6q22.1 // 23270 /// ENST00000368611 //TSP 3189580 ZBTB43 NM_014007 // NM_014007 −0.21 0.08 0.017 ZBTB43 //zinc finger and BTB domain containing 43 // 9q33-q34 // 2 3407926 CMASNM_018686 // NM_018686 −0.21 0.03 0.000 CMAS // cytidine monophosphateN- acetylneuraminic acid synthetase / 3249886 TET1 NM_030625 //NM_030625 −0.21 0.06 0.007 TET1 // tet oncogene 1 // 10q21 // 80312 ///ENST00000373644 // TET 3151970 MTSS1 NM_014751 // NM_014751 −0.21 0.070.009 MTSS1 // metastasis suppressor 1 // 8p22 // 9788 ///ENST0000032506 3937183 DGCR8 NM_022720 // NM_022720 −0.21 0.06 0.008DGCR8 // DiGeorge syndrome critical region gene 8 // 22q11.2 // 5443958253 C22orf28 BC016707 // BC016707 −0.22 0.08 0.019 C22orf28 //chromosome 22 open reading frame 28 // 22q12 // 51493 // 3607503 ABHD2NM_007011 // NM_007011 −0.22 0.07 0.010 ABHD2 // abhydrolase domaincontaining 2 // 15q26.1 // 11057 /// NM 2799030 SLC6A19 NM_001003841 //NM_001003841 −0.22 0.06 0.007 SLC6A19 // solute carrier family 6(neutral amino acid transport 3870611 LILRB3 NM_001081450 //NM_001081450 −0.22 0.08 0.016 LILRB3 // leukocyte immunoglobulin- likereceptor, subfamily B (w 3857811 C19orf12 NM_031448 // NM_031448 −0.220.08 0.019 C19orf12 // chromosome 19 open reading frame 12 // 19q12 //83636 / 2500667 FBLN7 NM_153214 // NM_153214 −0.22 0.08 0.019 FBLN7 //fibulin 7 // 2q13 // 129804 /// ENST00000331203 // FBLN7 / 3523156 TMTC4NM_032813 // NM_032813 −0.22 0.07 0.010 TMTC4 // transmembrane andtetratricopeptide repeat containing 4 // 2612371 EAF1 NM_033083 //NM_033083 −0.22 0.07 0.008 EAF1 // ELL associated factor 1 // 3p24.3 //85403 /// ENST00000396 3988638 LONRF3 NM_001031855 // NM_001031855 −0.230.08 0.012 LONRF3 // LON peptidase N- terminal domain and ring finger 3// X 3114240 C8orf32 BC008781 // BC008781 −0.23 0.08 0.016 C8orf32 //chromosome 8 open reading frame 32 // 8q24.13 // 55093 // 2460368 TTC13NM_024525 // NM_024525 −0.23 0.08 0.014 TTC13 // tetratricopeptiderepeat domain 13 // 1q42.2 // 79573 /// 2428425 PPM1J NM_005167 //NM_005167 −0.23 0.06 0.003 PPM1J // protein phosphatase 1J (PP2C domaincontaining) // 1p13.2 3194986 LCN12 NM_178536 // NM_178536 −0.23 0.060.004 LCN12 // lipocalin 12 // 9q34.3 // 286256 /// ENST00000371633 //LC 3642875 RAB11FIP3 NM_014700 // NM_014700 −0.23 0.07 0.010 RAB11FIP3// RAB11 family interacting protein 3 (class II) // 16p13 2532378 CHRNDNM_000751 // NM_000751 −0.23 0.08 0.018 CHRND // cholinergic receptor,nicotinic, delta // 2q33-q34 // 1144 2995667 ADCYAP1R1 NM_001118 //NM_001118 −0.23 0.05 0.002 ADCYAP1R1 // adenylate cyclase activatingpolypeptide 1 (pituitary) 3390641 ARHGAP20 NM_020809 // NM_020809 −0.230.05 0.003 ARHGAP20 // Rho GTPase activating protein 20 // 11q22.3-q23.1// 57 2830465 MYOT NM_006790 // NM_006790 −0.23 0.07 0.007 MYOT //myotilin // 5q31 // 9499 /// ENST00000239926 // MYOT // myo 2452069PIK3C2B NM_002646 // NM_002646 −0.23 0.02 0.000 PIK3C2B //phosphoinositide-3- kinase, class 2, beta polypeptide // 3744127 HES7NM_032580 // NM_032580 −0.23 0.09 0.019 HES7 // hairy and enhancer ofsplit 7 (Drosophila) // 17p13.1 // 84 3327057 FLJ14213 NM_024841 //NM_024841 −0.23 0.07 0.007 FLJ14213 // protor- 2 // 11p13-p12 // 79899/// ENST00000378867 // F 2664332 COLQ NM_005677 // NM_005677 −0.23 0.070.006 COLQ // collagen- like tail subunit (single strand of homotrimer)of 3829160 C19orf40 NM_152266 // NM_152266 −0.23 0.08 0.012 C19orf40 //chromosome 19 open reading frame 40 // 19q13.11 // 9144 3708798 SENP3NM_015670 // NM_015670 −0.23 0.06 0.005 SENP3 // SUMO1/sentrin/SMT3specific peptidase 3 // 17p13 // 26168 2358700 MGC29891 NM_144618 //NM_144618 −0.23 0.09 0.019 MGC29891 // hypothetical protein MGC29891 //1q21.2 // 126626 /// E 2755111 KLKB1 NM_000892 // NM_000892 −0.24 0.080.012 KLKB1 // kallikrein B, plasma (Fletcher factor) 1 // 4q34-q35 //38 2568968 UXS1 NM_025076 // NM_025076 −0.24 0.08 0.011 UXS1 // UDP-glucuronate decarboxylase 1 // 2q12.2 // 80146 /// BC00 2748923 GUCY1B3NM_000857 // NM_000857 −0.24 0.07 0.007 GUCY1B3 // guanylate cyclase 1,soluble, beta 3 // 4q31.3-q33 // 29 3816509 GADD45B NM_015675 //NM_015675 −0.24 0.09 0.016 GADD45B // growth arrest and DNA-damage-inducible, beta // 19p13.3 3376410 SLC22A24 BC034394 // BC034394 −0.240.07 0.007 SLC22A24 // solute carrier family 22, member 24 // 11q12.3 //283238 3286393 ZNF32 NM_006973 // NM_006973 −0.24 0.08 0.010 ZNF32 //zinc finger protein 32 // 10q22-q25 // 7580 /// NM_0010053 2540157 ODC1NM_002539 // NM_002539 −0.24 0.09 0.020 ODC1 // ornithine decarboxylase1 // 2p25 // 4953 /// ENST000002341 2994835 CHN2 NM_004067 // NM_004067−0.24 0.09 0.017 CHN2 // chimerin (chimaerin) 2 // 7p15.3 // 1124 ///NM_001039936 / 3603199 IDH3A NM_005530 // NM_005530 −0.24 0.05 0.001IDH3A // isocitrate dehydrogenase 3 (NAD+) alpha // 15q25.1-q25.2 /3040454 TWISTNB NM_001002926 // NM_001002926 −0.24 0.09 0.017 TWISTNB //TWIST neighbor // 7p15.3 // 221830 /// ENST0000022256 2497301 TMEM182NM_144632 // NM_144632 −0.24 0.07 0.007 TMEM182 // transmembrane protein182 // 2q12.1 // 130827 /// ENST00 3766716 TEX2 NM_018469 // NM_018469−0.25 0.07 0.007 TEX2 // testis expressed 2 // 17q23.3 // 55852 ///ENST00000258991 3458819 CYP27B1 NM_000785 // NM_000785 −0.25 0.08 0.009CYP27B1 // cytochrome P450, family 27, subfamily B, polypeptide 1 /3368940 ABTB2 NM_145804 // NM_145804 −0.25 0.08 0.010 ABTB2 // ankyrinrepeat and BTB (POZ) domain containing 2 // 11p13 3298924 MMRN2NM_024756 // NM_024756 −0.25 0.07 0.006 MMRN2 // multimerin 2 // 10q23.2// 79812 /// ENST00000372027 // MM 3529951 KIAA1305 NM_025081 //NM_025081 −0.25 0.08 0.011 KIAA1305 // KIAA1305 // 14q12 // 57523 ///BC008219 // KIAA1305 // 3006572 AUTS2 NM_015570 // NM_015570 −0.25 0.090.017 AUTS2 // autism susceptibility candidate 2 // 7q11.22 // 26053 ///3025500 BPGM NM_001724 // NM_001724 −0.25 0.10 0.018 BPGM // 2,3-bisphosphoglycerate mutase // 7q31-q34 // 669 /// NM_19 2494709 CNNM4NM_020184 // NM_020184 −0.26 0.09 0.016 CNNM4 // cyclin M4 // 2p12-p11.2// 26504 /// ENST00000377075 // CN 3329983 PTPRJ NM_002843 // NM_002843−0.26 0.08 0.010 PTPRJ // protein tyrosine phosphatase, receptor type, J// 11p11.2 2769346 LNX1 NM_032622 // NM_032622 −0.26 0.09 0.015 LNX1 //ligand of numb-protein X 1 // 4q12 // 84708 /// ENST0000030 3867195FAM83E NM_017708 // NM_017708 −0.26 0.09 0.013 FAM83E // family withsequence similarity 83, member E // 19q13.32- 3790529 GRP NM_002091 //NM_002091 −0.26 0.05 0.001 GRP // gastrin- releasing peptide //18q21.1-q21.32 // 2922 /// NM_0 3987029 TMEM164 NM_032227 // NM_032227−0.26 0.10 0.018 TMEM164 // transmembrane protein 164 // Xq22.3 // 84187/// ENST000 3526454 GRTP1 NM_024719 // NM_024719 −0.26 0.09 0.015 GRTP1// growth hormone regulated TBC protein 1 // 13q34 // 79774 / 2438344GPATCH4 NM_182679 // NM_182679 −0.26 0.07 0.006 GPATCH4 // G patchdomain containing 4 // 1q22 // 54865 /// NM_0155 3132927 NKX6-3NM_152568 // NM_152568 −0.27 0.09 0.014 NKX6-3 // NK6 homeobox 3 //8p11.21 // 157848 /// ENST00000343444 / 2672376 TESSP2 NM_182702 //NM_182702 −0.27 0.09 0.013 TESSP2 // testis serine protease 2 // 3p21.31// 339906 /// ENST000 2730347 C4orf35 NM_033122 // NM_033122 −0.27 0.100.019 C4orf35 // chromosome 4 open reading frame 35 // 4q13.3 // 85438// 3921068 ETS2 NM_005239 // NM_005239 −0.27 0.03 0.000 ETS2 // v-etserythroblastosis virus E26 oncogene homolog 2 (avian) 2532894 DGKDNM_152879 // NM_152879 −0.27 0.07 0.003 DGKD // diacylglycerol kinase,delta 130 kDa // 2q37.1 // 8527 /// N 4018454 AMOT NM_133265 //NM_133265 −0.27 0.09 0.012 AMOT // angiomotin // Xq23 // 154796 ///NM_001113490 // AMOT // an 3070507 RNF148 NM_198085 // NM_198085 −0.270.10 0.017 RNF148 // ring finger protein 148 // 7q31.33 // 378925 ///BC029264 3832256 SPINT2 NM_021102 // NM_021102 −0.27 0.10 0.017 SPINT2// serine peptidase inhibitor, Kunitz type, 2 // 19q13.1 // 3371225CHST1 NM_003654 // NM_003654 −0.27 0.07 0.005 CHST1 // carbohydrate(keratan sulfate Gal-6) sulfotransferase 1 // 3870494 TFPT NM_013342 //NM_013342 −0.27 0.09 0.010 TFPT // TCF3 (E2A) fusion partner (inchildhood Leukemia) // 19q13 3863811 PSG9 NM_002784 // NM_002784 −0.280.09 0.011 PSG9 // pregnancy specific beta-1- glycoprotein 9 // 19q13.2// 5678 3160175 VLDLR NM_003383 // NM_003383 −0.28 0.08 0.007 VLDLR //very low density lipoprotein receptor // 9p24 // 7436 /// 2794704 ASB5NM_080874 // NM_080874 −0.28 0.11 0.019 ASB5 // ankyrin repeat and SOCSbox-containing 5 // 4q34.2 // 14045 3908901 KCNB1 NM_004975 // NM_004975−0.28 0.09 0.009 KCNB1 // potassium voltage- gated channel, Shab-relatedsubfamily, m 3390852 FLJ45803 NM_207429 // NM_207429 −0.28 0.10 0.015FLJ45803 // FLJ45803 protein // 11q23.1 // 399948 /// ENST0000035542600689 EPHA4 NM_004438 // NM_004438 −0.29 0.07 0.003 EPHA4 // EPHreceptor A4 // 2q36.1 // 2043 /// ENST00000281821 // E 3469597 NUAK1NM_014840 // NM_014840 −0.29 0.09 0.009 NUAK1 // NUAK family, SNF1-likekinase, 1 // 12q23.3 // 9891 /// EN 3607232 ISG20L1 NM_022767 //NM_022767 −0.29 0.10 0.015 ISG20L1 // interferon stimulated exonucleasegene 20 kDa-like 1 // 1 2358426 ADAMTSL4 AK023606 // AK023606 −0.29 0.110.016 ADAMTSL4 // ADAMTS-like 4 // 1q21.2 // 54507 3853609 CYP4F2NM_001082 // NM_001082 −0.29 0.11 0.016 CYP4F2 // cytochrome P450,family 4, subfamily F, polypeptide 2 // 2936971 KIF25 NM_030615 //NM_030615 −0.30 0.09 0.008 KIF25 // kinesin family member 25 // 6q27 //3834 /// NM_005355 // 2997272 EEPD1 NM_030636 // NM_030636 −0.30 0.090.010 EEPD1 // endonuclease/exonuclease/ phosphatase family domaincontain 3961253 RPS19BP1 NM_194326 // NM_194326 −0.30 0.10 0.013RPS19BP1 // ribosomal protein S19 binding protein 1 // 22q13.1 // 93082373 VIPR2 NM_003382 // NM_003382 −0.30 0.10 0.011 VIPR2 //vasoactive intestinal peptide receptor 2 // 7q36.3 // 7434 2340961IL12RB2 NM_001559 // NM_001559 −0.30 0.08 0.005 IL12RB2 // interleukin12 receptor, beta 2 // 1p31.3-p31.2 // 3595 2736462 BMPR1B NM_001203 //NM_001203 −0.30 0.08 0.004 BMPR1B // bone morphogenetic proteinreceptor, type IB // 4q22-q24 3774504 — — −0.30 0.11 0.016 3395958 OR8B4NM_001005196 // NM_001005196 −0.30 0.11 0.018 OR8B4 // olfactoryreceptor, family 8, subfamily B, member 4 // 2806231 BXDC2 NM_018321 //NM_018321 −0.31 0.10 0.013 BXDC2 // brix domain containing 2 // 5p13.2// 55299 /// ENST000003 2396858 NPPB NM_002521 // NM_002521 −0.31 0.110.016 NPPB // natriuretic peptide precursor B // 1p36.2 // 4879 ///ENST0 3233322 C10orf18 NM_017782 // NM_017782 −0.31 0.06 0.001 C10orf18// chromosome 10 open reading frame 18 // 10p15.1 // 54906 2439101 FCRL1NM_052938 // NM_052938 −0.31 0.06 0.001 FCRL1 // Fc receptor-like 1 //1q21-q22 // 115350 /// ENST000003681 2413907 DHCR24 NM_014762 //NM_014762 −0.31 0.11 0.014 DHCR24 // 24- dehydrocholesterol reductase //1p33-p31.1 // 1718 /// 3231186 C9orf37 NM_032937 // NM_032937 −0.31 0.090.008 C9orf37 // chromosome 9 open reading frame 37 // 9q34.3 // 85026// 2669955 XIRP1 NM_194293 // NM_194293 −0.32 0.11 0.013 XIRP1 // xinactin- binding repeat containing 1 // 3p22.2 // 165904 3345222 AMOTL1NM_130847 // NM_130847 −0.32 0.11 0.012 AMOTL1 // angiomotin like 1 //11q14.3 // 154810 /// ENST0000031782 2573326 FLJ14816 BC112205 //BC112205 −0.32 0.11 0.016 FLJ14816 // hypothetical protein FLJ14816 //2q14.2 // 84931 /// BC1 3349437 UNQ2550 AY358815 // AY358815 −0.32 0.090.005 UNQ2550 // SFVP2550 // 11q23.1 // 100130653 3951117 ACR NM_001097// NM_001097 −0.32 0.12 0.017 ACR // acrosin // 22q13-qter| 22q13.33 //49 /// ENST00000216139 // 2489140 — — −0.32 0.07 0.002 2562115 LSM3CR457185 // LSM3 CR457185 −0.32 0.11 0.011 // LSM3 homolog, U6 smallnuclear RNA associated (S. cerevisiae 3572975 NGB NM_021257 // NM_021257−0.33 0.09 0.004 NGB // neuroglobin // 14q24.3 // 58157 ///ENST00000298352 // NGB / 2439350 OR6N1 NM_001005185 // NM_001005185−0.33 0.10 0.009 OR6N1 // olfactory receptor, family 6, subfamily N,member 1 // 3590275 CHAC1 NM_024111 // NM_024111 −0.33 0.12 0.014 CHAC1// ChaC, cation transport regulator homolog 1 (E. coli) // 15 2397898HSPB7 NM_014424 // NM_014424 −0.33 0.12 0.015 HSPB7 // heat shock 27 kDaprotein family, member 7 (cardiovascular) 2364677 PBX1 NM_002585 //NM_002585 −0.34 0.07 0.001 PBX1 // pre-B-cell leukemia homeobox 1 //1q23 // 5087 /// ENST0000 2474409 DNAJC5G NM_173650 // NM_173650 −0.340.09 0.004 DNAJC5G // DnaJ (Hsp40) homolog, subfamily C, member 5 gamma// 2p2 3581373 — — −0.34 0.12 0.014 3508330 HSPH1 NM_006644 // NM_006644−0.34 0.13 0.019 HSPH1 // heat shock 105 kDa/110 kDa protein 1 //13q12.3 // 10808 /// 3751164 DHRS13 NM_144683 // NM_144683 −0.35 0.100.006 DHRS13 // dehydrogenase/reductase (SDR family) member 13 //17q11.2 2908179 VEGFA NM_001025366 // NM_001025366 −0.35 0.13 0.016VEGFA // vascular endothelial growth factor A // 6p12 // 7422 // 3962448dJ222E13.2 NR_002184 // NR_002184 −0.35 0.12 0.014 dJ222E13.2 // similarto CGI-96 // 22q13.2 // 91695 /// BC073834 // 3747638 LOC201164 BC031263// BC031263 −0.35 0.09 0.004 LOC201164 // similar to CG12314 geneproduct // 17p11.2 // 201164 // 2821981 TMEM157 NM_198507 // NM_198507−0.35 0.12 0.015 TMEM157 // transmembrane protein 157 // 5q21.1 //345757 /// ENST00 3123675 PPP1R3B NM_024607 // NM_024607 −0.35 0.120.014 PPP1R3B // protein phosphatase 1, regulatory (inhibitor) subunit3B 2656837 ST6GAL1 NM_173216 // NM_173216 −0.35 0.13 0.016 ST6GAL1 //ST6 beta-galactosamide alpha-2,6- sialyltranferase 1 // 3 3746574 PMP22NM_000304 // NM_000304 −0.36 0.09 0.004 PMP22 // peripheral myelinprotein 22 // 17p12-p11.2 // 5376 /// NM 2771342 EPHA5 NM_004439 //NM_004439 −0.36 0.09 0.003 EPHA5 // EPH receptor A5 // 4q13.1 // 2044/// NM_182472 // EPHA5 / 2888674 MXD3 NM_031300 // NM_031300 −0.36 0.120.012 MXD3 // MAX dimerization protein 3 // 5q35.3 // 83463 ///ENST00000 2353477 ATP1A1 NM_000701 // NM_000701 −0.36 0.11 0.007 ATP1A1// ATPase, Na+/K+ transporting, alpha 1 polypeptide // 1p21 3956984ZMAT5 NM_019103 // NM_019103 −0.36 0.11 0.009 ZMAT5 // zinc finger,matrin type 5 // 22cen-q12.3 // 55954 /// NM_(—) 2551651 ATP6V1E2NM_080653 // NM_080653 −0.37 0.13 0.017 ATP6V1E2 // ATPase, H+transporting, lysosomal 31 kDa, V1 subunit E2 3578069 C14orf139 BC008299// BC008299 −0.37 0.13 0.016 C14orf139 // chromosome 14 open readingframe 139 // 14q32.13 // 796 2428501 SLC16A1 NM_003051 // NM_003051−0.37 0.14 0.018 SLC16A1 // solute carrier family 16, member 1(monocarboxylic acid 3061621 TFPI2 NM_006528 // NM_006528 −0.37 0.090.002 TFPI2 // tissue factor pathway inhibitor 2 // 7q22 // 7980 ///ENST 3705516 LOC100131454 AF229804 // AF229804 −0.38 0.11 0.008LOC100131454 // similar to hCG1646635 // 17p13.3 // 100131454 /// EN3306299 XPNPEP1 NM_020383 // NM_020383 −0.38 0.14 0.018 XPNPEP1 // X-prolyl aminopeptidase (aminopeptidase P) 1, soluble // 2763550 PPARGC1ANM_013261 // NM_013261 −0.38 0.13 0.012 PPARGC1A // peroxisomeproliferator- activated receptor gamma, coact 2769063 USP46 NM_022832 //NM_022832 −0.38 0.13 0.013 USP46 // ubiquitin specific peptidase 46 //4q12 // 64854 /// ENST0 3806459 ST8SIA5 NM_013305 // NM_013305 −0.380.10 0.004 ST8SIA5 // ST8 alpha-N-acetyl- neuraminide alpha-2,8-sialyltransfera 3190151 SLC25A25 NM_001006641 // NM_001006641 −0.390.09 0.003 SLC25A25 // solute carrier family 25 (mitochondrial carrier;pho 2489172 MTHFD2 NM_001040409 // NM_001040409 −0.39 0.05 0.000 MTHFD2// methylenetetrahydrofolate dehydrogenase (NADP+ depende 2952065 PPIL1NM_016059 // NM_016059 −0.39 0.10 0.005 PPIL1 // peptidylprolylisomerase (cyclophilin)-like 1 // 6p21.1 // 3382015 CHRDL2 NM_015424 //NM_015424 −0.39 0.10 0.003 CHRDL2 // chordin-like 2 // 11q14 // 25884/// ENST00000263671 // C 2711139 ATP13A5 NM_198505 // NM_198505 −0.400.11 0.005 ATP13A5 // ATPase type 13A5 // 3q29 // 344905 ///ENST00000342358 / 2633917 RG9MTD1 NM_017819 // NM_017819 −0.41 0.140.013 RG9MTD1 // RNA (guanine-9-) methyltransferase domain containing 1/ 2974671 C6orf192 NM_052831 // NM_052831 −0.41 0.15 0.018 C6orf192 //chromosome 6 open reading frame 192 // 6q22.3-q23.3 // 2982270 FLJ27255ENST00000355047 ENST00000355047 −0.41 0.12 0.007 // FLJ27255 //hypothetical LOC401281 // 6q25.3 // 401281 /// AK 2778273 PGDS NM_014485// NM_014485 −0.41 0.08 0.001 PGDS // prostaglandin D2 synthase,hematopoietic // 4q22.3 // 27306 3005332 RCP9 NM_014478 // NM_014478−0.41 0.14 0.013 RCP9 // calcitonin gene-related peptide-receptorcomponent protein 2650393 PPM1L NM_139245 // NM_139245 −0.42 0.12 0.006PPM1L // protein phosphatase 1 (formerly 2C)-like // 3q26.1 // 15173463056 CSRP2 NM_001321 // NM_001321 −0.42 0.11 0.005 CSRP2 // cysteineand glycine-rich protein 2 // 12q21.1 // 1466 /// 2459405 — — −0.43 0.100.003 2570238 NPHP1 NM_000272 // NM_000272 −0.43 0.06 0.000 NPHP1 //nephronophthisis 1 (juvenile) // 2q13 // 4867 /// NM_20718 2840616 NPM1NM_002520 // NM_002520 −0.43 0.14 0.010 NPM1 // nucleophosmin (nucleolarphosphoprotein B23, numatrin) // 5 3601051 NEO1 NM_002499 // NM_002499−0.43 0.09 0.002 NEO1 // neogenin homolog 1 (chicken) // 15q22.3-q23 //4756 /// ENS 3936515 TUBA8 NM_018943 // NM_018943 −0.43 0.10 0.002 TUBA8// tubulin, alpha 8 // 22q11.1 // 51807 /// ENST00000330423 / 2725013UCHL1 NM_004181 // NM_004181 −0.44 0.11 0.004 UCHL1 // ubiquitincarboxyl-terminal esterase L1 (ubiquitin thioles 2380590 TGFB2 NM_003238// NM_003238 −0.44 0.16 0.017 TGFB2 // transforming growth factor, beta2 // 1q41 // 7042 /// ENS 2496382 NPAS2 NM_002518 // NM_002518 −0.460.10 0.002 NPAS2 // neuronal PAS domain protein 2 // 2q11.2 // 4862 ///ENST00 3841574 LILRB1 NM_006669 // NM_006669 −0.46 0.16 0.015 LILRB1 //leukocyte immunoglobulin- like receptor, subfamily B (with 3726960 NME2NM_001018137 // NM_001018137 −0.47 0.16 0.013 NME2 // non- metastaticcells 2, protein (NM23B) expressed in // 2649367 PTX3 NM_002852 //NM_002852 −0.47 0.11 0.002 PTX3 // pentraxin- related gene, rapidlyinduced by IL-1 beta // 3q2 2909483 GPR111 NM_153839 // NM_153839 −0.470.13 0.006 GPR111 // G protein-coupled receptor 111 // 6p12.3 // 222611/// EN 2881950 SLC36A2 NM_181776 // NM_181776 −0.48 0.12 0.004 SLC36A2// solute carrier family 36 (proton/amino acid symporter), 3441190 FGF6NM_020996 // NM_020996 −0.48 0.12 0.004 FGF6 // fibroblast growth factor6 // 12p13 // 2251 /// ENST0000022 3028911 C7orf34 NM_178829 //NM_178829 −0.49 0.18 0.019 C7orf34 // chromosome 7 open reading frame 34// 7q34 // 135927 /// 2830861 EGR1 NM_001964 // NM_001964 −0.49 0.190.020 EGR1 // early growth response 1 // 5q31.1 // 1958 ///ENST000002399 3323891 GAS2 NM_177553 // NM_177553 −0.49 0.16 0.011 GAS2// growth arrest-specific 2 // 11p14.3-p15.2 // 2620 /// NM_00 2497252SLC9A2 NM_003048 // NM_003048 −0.50 0.11 0.002 SLC9A2 // solute carrierfamily 9 (sodium/hydrogen exchanger), memb 3018484 GPR22 NM_005295 //NM_005295 −0.51 0.15 0.008 GPR22 // G protein-coupled receptor 22 //7q22-q31.1 // 2845 /// EN 2712632 TFRC NM_003234 // NM_003234 −0.51 0.120.003 TFRC // transferrin receptor (p90, CD71) // 3q29 // 7037 ///ENST00 3214451 NFIL3 NM_005384 // NM_005384 −0.53 0.14 0.004 NFIL3 //nuclear factor, interleukin 3 regulated // 9q22 // 4783 // 2435981S100A12 NM_005621 // NM_005621 −0.54 0.19 0.014 S100A12 // S100 calciumbinding protein A12 // 1q21 // 6283 /// ENS 3320675 RIG U32331 // RIG //U32331 −0.54 0.10 0.001 regulated in glioma // 11p15.1 // 10530 3290746SLC16A9 NM_194298 // NM_194298 −0.54 0.15 0.006 SLC16A9 // solutecarrier family 16, member 9 (monocarboxylic acid 3055703 NSUN5CNM_032158 // NM_032158 −0.57 0.17 0.008 NSUN5C // NOL1/NOP2/Sun domainfamily, member 5C // 7q11.23 // 2602 3265494 TRUB1 NM_139169 //NM_139169 −0.57 0.17 0.008 TRUB1 // TruB pseudouridine (psi) synthasehomolog 1 (E. coli) // 1 3374213 OR1S2 NM_001004459 // NM_001004459−0.58 0.20 0.013 OR1S2 // olfactory receptor, family 1, subfamily S,member 2 // 3318253 OR51L1 NM_001004755 // NM_001004755 −0.59 0.18 0.009OR51L1 // olfactory receptor, family 51, subfamily L, member 1 / 3294280DNAJC9 NM_015190 // NM_015190 −0.59 0.22 0.018 DNAJC9 // DnaJ (Hsp40)homolog, subfamily C, member 9 // 10q22.2 // 2899095 HIST1H4A NM_003538// NM_003538 −0.60 0.16 0.005 HIST1H4A // histone cluster 1, H4a //6p21.3 // 8359 /// ENST000003 2378068 G0S2 NM_015714 // NM_015714 −0.630.22 0.016 G0S2 // G0/G1switch 2 // 1q32.2-q41 // 50486 ///ENST00000367029 // 3737677 LOC100129503 AF218021 // AF218021 −0.64 0.190.007 LOC100129503 // hypothetical protein LOC100129503 // 17q25.3 //1001 3300115 PPP1R3C NM_005398 // NM_005398 −0.69 0.26 0.020 PPP1R3C //protein phosphatase 1, regulatory (inhibitor) subunit 3C 3279058 ACBD7NM_001039844 // NM_001039844 −0.69 0.13 0.001 ACBD7 // acyl- Coenzyme Abinding domain containing 7 // 10p13 // 4031156 RPS4Y2 NM_001039567 //NM_001039567 −0.71 0.17 0.003 RPS4Y2 // ribosomal protein S4, Y-linked 2// Yq11.223 // 140032 2979246 RAET1L NM_130900 // NM_130900 −0.75 0.260.013 RAET1L // retinoic acid early transcript 1L // 6q25.1 // 154064/// 3321150 ARNTL NM_001178 // NM_001178 −0.80 0.20 0.004 ARNTL // arylhydrocarbon receptor nuclear translocator-like // 11p 3862873 CYP2A6NM_000762 // NM_000762 −1.12 0.34 0.009 CYP2A6 // cytochrome P450,family 2, subfamily A, polypeptide 6 //

4. Identification of Ursolic Acid as an Inhibitor of Fasting-InducedMuscle Atrophy.

The Connectivity Map describes the effects of >1300 bioactive smallmolecules on global mRNA expression in several cultured cell lines, andcontains search algorithms that permit comparisons betweencompound-specific mRNA expression signatures and mRNA expressionsignatures of interest (Lamb J, et al. (2006) Science (New York, N.Y.313(5795): 1929-1935). It was hypothesized herein that querying theConnectivity Map with the mRNA expression signature of fasting (muscleatrophy signature-1) would identify inhibitors of atrophy-associatedgene expression and thus, potential inhibitors of muscle atrophy. It wasalso reasoned herein that increasing the specificity of the query wouldenhance the output. To this end, as described herein, an evolutionarilyconserved mRNA expression signature of fasting was discovered bycomparing the effect of fasting on human skeletal muscle to the effectof a 24 h fast on mouse skeletal muscle. The mouse studies weredescribed previously (Ebert S M, et al. (2010) Molecular endocrinology24(4):790-799). Altogether, 35 mRNAs that were increased by fasting and40 mRNAs that were decreased by fasting were identified in both humanand mouse skeletal muscle (Table X2; the data in column labeled “Change”show mean changes in log 2 hybridization signals between fasting and fedstates for the species indicated, [Mean log 2 mRNA levels for fasted]minus [Mean log 2 mRNA levels in unfasted]; P-values were determinedwith paired t-tests). The data shown in Table X2 includes all mRNAswhose levels were increased by fasting in human muscle (P≤0.02) and inmouse muscle (P≤0.05), and all mRNAs whose levels were decreased byfasting in human muscle (P≤0.02) and in mouse muscle (P≤0.05). Of themRNAs shown in Table X2, 63 mRNAs were represented on the HG-U133Aarrays used in the Connectivity Map (FIG. 6A). These mRNAs (31 increasedby fasting and 32 decreased by fasting) were used to query theConnectivity Map for candidate small molecule inhibitors of muscleatrophy.

TABLE X2 Fasting-regulated mRNAs common to human and mouse skeletalmuscle. Human Mouse Mean Log2 Change Mean Log2 Change mRNA Protein(Fasting - Fed) P (Fasting - Fed) P PDK4 pyruvate dehydrogenase kinase,isozyme 4 2.15 0.000 1.91 0.000 TXNIP thioredoxin interacting protein0.85 0.004 0.60 0.038 FBXO32 F-box protein 32 0.82 0.002 2.13 0.000SLC38A2 solute carrier family 38, member 2 0.62 0.001 0.33 0.036 UCP3uncoupling protein 3 (mitochondrial, proton carrier) 0.59 0.000 1.020.001 ZFAND5 zinc finger, AN1-type domain 5 0.51 0.005 0.57 0.001 HMOX1heme oxygenase (decycling) 1 0.46 0.006 0.17 0.035 SESN1 sestrin 1 0.460.004 1.51 0.001 GABARAPL1 GABA(A) receptor-associated protein like 10.39 0.004 1.18 0.000 CAT catalase 0.39 0.003 0.85 0.001 CITED2Cbp/p300-interacting transactivator, with Glu/Asp-rich 0.37 0.005 0.290.010 carboxy- terminal domain ABCA1 ATP-binding cassette, sub-family A(ABC1), member 1 0.37 0.016 0.26 0.018 FBXL20 F-box and leucine-richrepeat protein 20 0.35 0.002 0.46 0.001 XPO4 exportin 4 0.31 0.009 0.220.022 HERPUD1 homocysteine-inducible, endoplasmic reticulum stress- 0.290.003 0.27 0.029 inducible, ubiquitin-like domain 1 ACOX1 acyl-CoenzymeA oxidase 1, palmitoyl 0.29 0.013 0.53 0.006 NOX4 NADPH oxidase 4 0.280.002 0.41 0.018 UBE4A ubiquitination factor E4A (UFD2 homolog, yeast)0.27 0.004 1.08 0.010 INSR insulin receptor 0.24 0.014 0.58 0.003 IGF1Rinsulin-like growth factor 1 receptor 0.23 0.013 0.40 0.001 PANK1pantothenate kinase 1 0.21 0.007 0.78 0.000 NBR1 neighbor of BRCA1 gene1 0.21 0.017 0.39 0.009 RORA RAR-related orphan receptor A 0.21 0.0060.39 0.006 TMEM71 transmembrane protein 71 0.21 0.009 0.40 0.008 CPT1Acarnitine palmitoyltransferase 1A (liver) 0.21 0.001 0.21 0.020 UCP2uncoupling protein 2 (mitochondrial, proton carrier) 0.20 0.005 0.330.024 TULP3 tubby like protein 3 0.19 0.008 0.22 0.008 MED13L mediatorcomplex subunit 13-like 0.18 0.000 0.23 0.011 CALCOCO1 calcium bindingand coiled coil domain 1 0.16 0.010 0.31 0.028 MYO5A myosin VA (heavychain 12, myoxin) 0.14 0.006 0.36 0.012 PPAP2B phosphatidic acidphosphatase type 2B 0.13 0.007 0.09 0.029 SRRM2 serine/argininerepetitive matrix 2 0.13 0.007 0.24 0.040 ADPGK ADP-dependentglucokinase 0.13 0.007 0.16 0.009 SUPT6H suppressor of Ty 6 homolog (S.cerevisiae) 0.11 0.005 0.26 0.036 SFRS8 splicing factor,arginine/serine-rich 8 0.08 0.016 0.13 0.011 NFYA nuclear transcriptionfactor Y, alpha −0.07 0.011 −0.31 0.045 MRPS15 mitochondrial ribosomalprotein S15 −0.11 0.003 −0.25 0.001 PDE7B phosphodiesterase 7B −0.120.013 −0.51 0.011 WDR1 WD repeat domain 1 −0.14 0.019 −0.21 0.047 ACACAacetyl-Coenzyme A carboxylase alpha −0.15 0.010 −0.22 0.041 AXIN2 axin 2(conductin, axil) −0.15 0.013 −0.12 0.046 CASQ1 calsequestrin 1(fast-twitch, skeletal muscle) −0.16 0.015 −0.26 0.015 ZNF280B zincfinger protein 280B −0.16 0.005 −0.34 0.046 JTB jumping translocationbreakpoint −0.16 0.014 −0.42 0.030 CACNB1 calcium channel,voltage-dependent, beta 1 subunit −0.17 0.013 −0.43 0.003 ALG2asparagine-linked glycosylation 2 homolog −0.17 0.011 −0.39 0.019TSPAN13 tetraspanin 13 −0.18 0.006 −0.30 0.028 P4HA2procollagen-proline, 2-oxoglutarate 4-dioxygenase, alpha −0.18 0.007−0.12 0.012 II polypeptide TTLL1 tubulin tyrosine ligase-like family,member 1 −0.18 0.001 −0.29 0.043 SUV39H2 suppressor of variegation 3-9homolog 2 (Drosophila) −0.20 0.011 −0.26 0.014 SLC4A4 solute carrierfamily 4, sodium bicarbonate cotransporter, −0.20 0.007 −0.69 0.003member 4 DNMT3A DNA (cytosine-5-)-methyltransferase 3 alpha −0.20 0.007−0.48 0.000 FEZ2 fasciculation and elongation protein zeta 2 (zygin II)−0.21 0.000 −0.50 0.019 MTSS1 metastasis suppressor 1 −0.21 0.009 −0.220.033 TMTC4 transmembrane and tetratricopeptide repeat containing 4−0.22 0.010 −0.17 0.035 PPM1J protein phosphatase 1J (PP2C domaincontaining) −0.23 0.003 −0.30 0.012 ARHGAP20 Rho GTPase activatingprotein 20 −0.23 0.003 −0.22 0.013 ABTB2 ankyrin repeat and BTB (POZ)domain containing 2 −0.25 0.010 −0.18 0.005 CNNM4 cyclin M4 −0.26 0.016−0.27 0.005 GRTP1 growth hormone regulated TBC protein 1 −0.26 0.015−0.54 0.002 RNF148 ring finger protein 148 −0.27 0.017 −0.35 0.014SPINT2 serine peptidase inhibitor, Kunitz type, 2 −0.27 0.017 −0.230.026 PBX1 pre-B-cell leukemia homeobox 1 −0.34 0.001 −0.22 0.000 HSPH1heat shock 105 kDa/110 kDa protein 1 −0.34 0.019 −0.20 0.043 VEGFAvascular endothelial growth factor A −0.35 0.016 −0.26 0.002 PMP22peripheral myelin protein 22 −0.36 0.004 −0.13 0.012 PPARGC1A peroxisomeproliferative activated receptor, −0.38 0.012 −0.39 0.030 gamma,coactivator 1 alpha ST8SIA5 ST8 alpha-N-acetyl-neuraminide alpha-2,8-−0.38 0.004 −0.48 0.011 sialyltransferase 5 PPIL1 peptidylprolylisomerase (cyclophilin)-like 1 −0.39 0.005 −0.52 0.016 PPM1L proteinphosphatase 1 (formerly 2C)-like −0.42 0.006 −0.46 0.000 NEO1 neogeninhomolog 1 (chicken) −0.43 0.002 −0.31 0.037 TGFB2 transforming growthfactor, beta 2 −0.44 0.017 −0.30 0.003 PTX3 pentraxin-related gene,rapidly induced by IL-1 beta −0.47 0.002 −0.48 0.000 GAS2 growtharrest-specific 2 −0.49 0.011 −0.23 0.044 TFRC transferrin receptor(p90, CD71) −0.51 0.003 −1.37 0.011

The left side of FIG. 6B shows the 10 Connectivity Map instances (ordata sets) with the most significant positive correlations (P<0.004) tothe effect of fasting in skeletal muscle. The connectivity score,represented on the y-axis, is a measure of the strength of thecorrelation (Lamb J, et al. (2006) Science (New York, N.Y.313(5795):1929-1935); the compound and cell-line is shown below the barrepresenting the Connectivity Score. Of these, 6 involved wortmannin orLY-294002 (inhibitors of phosphoinositide 3-kinase (PI3K)) or rapamycin(an inhibitor of the mammalian target of rapamycin complex 1 (mTORC1)).Since PI3K and mTORC1 mediate effects of insulin and IGF-I, and sinceinsulin/IGF-I signaling inhibits muscle atrophy and atrophy-associatedchanges in skeletal muscle mRNA expression (Bodine S C, et al. (2001)Nat Cell Biol 3(11):1014-1019; Sandri M, et al. (2004) Cell117(3):399-412), these results lent confidence that the Connectivity Mapmight be used to identify potential inhibitors of muscle atrophy. Theright side of FIG. 6B shows the 10 Connectivity Map instances with themost significant negative correlations (P<0.004) to the effect offasting in skeletal muscle. These compounds, whose effects on culturedcell lines were opposite to the effect of fasting on muscle, includedmetformin (an insulin-sensitizing agent widely used to treat type 2diabetes), as well as ursolic acid. Further experiments focused onmetformin and ursolic acid. To test the hypothesis that metformin andursolic acid might reduce fasting-induced muscle atrophy, each compoundwas administered, or vehicle alone, via i.p. injection to C57BL/6 mice.The mice were then fasted, and after 12 hours of fasting, the micereceived a second dose of the compound or vehicle. After 24 hours offasting, the blood glucose was measured and muscles were harvested. Thedata shown in FIGS. 4C-4H are means±SEM from 16 mice. Both metformin(250 mg/kg) and ursolic acid (200 mg/kg) significantly reduced fastingblood glucose (FIGS. 4C and 4D). The effects of metformin and ursolicacid on fasting-induced muscle atrophy were also examined, i.e. theeffect of 24 h fast (relative to ad lib feeding) on wet weight of lowerhindlimb skeletal muscle (bilateral tibialis anterior (“TA” muscle),gastrocnemius, and soleus; see FIGS. 4E-4G). In the absence of metforminand ursolic acid, fasting reduced muscle weight by 9% (FIG. 6E).Although metformin did not alter muscle weight in fasted mice (FIG. 6F),ursolic acid increased it by 7±2% (FIG. 6G). Moreover, consistent withthe predicted inhibitory effect on fasting-induced gene expressiondescribed herein, ursolic acid reduced fasting levels of atrogin-1 andMuRF1 mRNA levels in the TA muscles of fasted mice (FIG. 6H; the datashown are normalized to the levels in vehicle-treated mice, which wereset at 1). In FIGS. 4E-4H, each data point represents one mouse and thehorizontal bars denote the means. In FIGS. 4C-4H, P-values weredetermined using unpaired t-tests. Thus, ursolic acid, but notmetformin, decreased fasting-induced muscle atrophy.

5. Ursolic Acid Reduces Denervation-Induced Muscle Atrophy.

The Connectivity Map was queried with a second mRNA expressionsignature, muscle atrophy signature-2 (described above), to determine ifthis muscle atrophy signature would also correlate with ursolic acid,among other compounds. As described above, muscle atrophy signature-2was an mRNA expression signature identified as described herein forhuman skeletal muscle mRNAs that were induced or repressed by fastingand also by spinal cord injury (“SCI”). The studies of the effects ofSCI on human skeletal muscle gene expression were described previously(Adams C M, et al. (2011) Muscle Nerve. 43(1):65-75). Using thisapproach with the muscle atrophy expression signatures described herein,there were 18 human mRNAs that were increased by fasting and SCI, and 17human mRNAs that were decreased by fasting and SCI, and are shown inTable X3 (“Change” represents mean changes in log 2 hybridizationsignals for pairs as indicated, e.g. fasting and fed states for columnlabeled “(Fasting—Fed)” or untrained and trained for the column labeled“(Untrained—Trained)”). The data in Table X3 include all mRNAs whoselevels were increased by fasting (P≤0.02) and by SCI (P≤0.05), and allmRNAs whose levels were decreased by fasting (P≤0.02) and by SCI(P≤0.05). P-values in Table X3 were determined with paired t-tests.

TABLE X3 Human skeletal muscle mRNAs induced or repressed by fasting andSCI. EFFECT OF FASTING EFFECT OF SCI Change Change mRNA Protein(Fasting - Fed) P (Untrained - Trained) P OR1D4 olfactory receptor,family 1, subfamily D, member 4 0.50 0.019 0.65 0.030 RHOBTB1Rho-related BTB domain containing 1 0.48 0.001 0.71 0.032 TSPAN8tetraspanin 8 0.39 0.015 1.79 0.023 FLJ33996 hypothetical proteinFLJ33996 0.39 0.019 0.68 0.020 NUPR1 nuclear protein 1 0.35 0.007 0.650.030 IRS2 insulin receptor substrate 2 0.34 0.004 0.21 0.035 NPC2Niemann-Pick disease, type C2 0.30 0.011 0.39 0.042 KLF11 Kruppel-likefactor 11 0.29 0.011 0.22 0.034 ZNF682 zinc finger protein 682 0.280.017 0.72 0.013 NOX4 NADPH oxidase 4 0.28 0.002 0.56 0.007 PLXDC2plexin domain containing 2 0.26 0.013 0.38 0.022 CTDSP2 CTD smallphosphatase 2 0.25 0.003 0.34 0.021 CAV3 caveolin 3 0.24 0.007 0.560.020 IGF1R insulin-like growth factor 1 receptor 0.23 0.013 0.63 0.040FLJ14154 hypothetical protein FLJ14154 0.22 0.005 0.30 0.021 CUGBP2 CUGtriplet repeat, RNA binding protein 2 0.21 0.004 0.14 0.034 MLLmyeloid/lymphoid or mixed-lineage leukemia 0.14 0.016 0.30 0.040 SUPT6Hsuppressor of Ty 6 homolog 0.11 0.005 0.19 0.024 MRPS15 mitochondrialribosomal protein S15 −0.11 0.003 −0.33 0.001 RFXDC2 regulatory factor Xdomain containing 2 −0.12 0.012 −0.10 0.037 PDE7B phosphodiesterase 7B−0.12 0.013 −0.39 0.011 PFDN6 prefoldin subunit 6 −0.14 0.014 −0.420.021 ZNF280B zinc finger protein 280B −0.16 0.005 −0.30 0.028 TSPAN13tetraspanin 13 −0.18 0.006 −0.56 0.023 TTLL1 tubulin tyrosineligase-like family, member 1 −0.18 0.001 −0.37 0.020 CMAS cytidinemonophosphate N-acetylneuraminic acid synthetase −0.21 0.000 −0.22 0.025C8orf32 chromosome 8 open reading frame 32 −0.23 0.016 −0.11 0.049GUCY1B3 guanylate cyclase 1, soluble, beta 3 −0.24 0.007 −0.24 0.008ZNF32 zinc finger protein 32 −0.24 0.010 −0.21 0.030 VLDLR very lowdensity lipoprotein receptor −0.28 0.007 −0.16 0.015 HSPB7 heat shock 27kDa protein family, member 7 (cardiovascular) −0.33 0.015 −0.77 0.032VEGFA vascular endothelial growth factor A −0.35 0.016 −0.43 0.020SLC16A1 solute carrier family 16, member 1 −0.37 0.018 −0.94 0.015PPARGC1A peroxisome proliferative activated receptor, gamma, coactivator1 alpha −0.38 0.012 −0.74 0.001 C6orf192 chromosome 6 open reading frame192 −0.41 0.018 −0.39 0.042

Of the mRNAs listed in Table X3, 29 were represented on the HG-U133Aarrays used in the Connectivity Map (FIG. 7A), but only 10 were commonto the 63 mRNAs used in the first Connectivity Map query described abovefor muscle atrophy signature-1 (IGF-IR, NOX4, SUPT6H, MRPS15, PDE7B,PGC-1a, TSPAN13, TTLL1, VEGFA and ZNF280B). The mRNAs listed in FIG. 7Arepresent human muscle atrophy signature-2: mRNAs altered by bothfasting and SCI in human muscle. These mRNAs, as described above, wereused to query the Connectivity Map. Inclusion criteria were: P≤0.02 infasted human muscle (by t-test), P≤0.05 in untrained, paralyzed muscle(by t-test), and the existence of complimentary probes on HG-U133Aarrays. Connectivity Map instances with the most significant positiveand negative correlations to the effect of fasting and SCI in humanmuscle. P<0.005 for all compounds are shown in FIG. 7B. The resultspartially overlapped with the results of the first search: both searchstrategies identified LY-294002, wortmannin and rapamycin as predictedmimics of atrophy-inducing stress, and ursolic acid (but not metformin)as a predicted inhibitor (FIG. 7B).

Because muscle atrophy signature-2 utilized data from SCI subjects, itwas hypothesized that ursolic acid might reduce denervation-inducedmuscle atrophy. To test this, the left hindlimb muscles adenervation-induced skeletal muscle atrophy model in mouse was used.Briefly, on day 0, the left hindlimbs of C57BL/6 mice were denervated bytranssecting the left sciatic nerve. This approach allowed the righthindlimb to serve as an intra-subject control. Mice were thenadministered ursolic acid (200 mg/kg) or an equivalent volume of vehiclealone (corn oil) via i.p. injection twice daily for seven days. Duringthis time, mice continued to have ad libitum access to food. On day 7,muscle tissues were harvested for analysis, and the left (denervated)and right (innervated) hindlimb muscles in both groups (ursolic acid vs.vehicle administration) were compared. Ursolic acid significantlydecreased denervation-induced muscle loss (FIG. 7C). In FIG. 7C, weightsof the left (denervated) lower hindlimb muscles were normalized toweights of the right (innervated) lower hindlimb muscles from the samemouse. Each data point represents one mouse, and horizontal bars denotethe means and the P-value was determined using an unpaired t-test.Histologically, this effect of ursolic acid was reflected as an increasein the size of denervated skeletal muscle fiber diameter in denervatedgastrocnemius (D) and TA (E) muscles (FIGS. 5D and 5E, respectively).The data shown in FIGS. 5D and 5E are from >2500 muscle fibers percondition; P<0.0001 by unpaired t-test. Thus, ursolic acid reduceddenervation-induced muscle atrophy.

6. Ursolic Acid Induces Skeletal Muscle Hypertrophy.

The results from the denervation-induced muscle atrophy model suggestedthat ursolic acid reduced muscle atrophy, thus the hypothesis thatursolic acid might promote muscle hypertrophy in the absence of anatrophy-inducing stress was reasonable. Mice were provided ad lib accessto either standard chow (control diet) or standard chow supplementedwith 0.27% ursolic acid (ursolic acid diet) for 5 weeks before gripstrength was measured and tissues were harvested. After five weeks, miceadministered ursolic had increased lower hindlimb muscle weight (FIG.8A), quadriceps weight (FIG. 8B), and upper forelimb muscle (triceps andbiceps) weight (FIG. 8C). Each data point in FIGS. 6A-6C represents onemouse, and horizontal bars denote the means. The effect of ursolic acidin this study on skeletal muscle fiber size distribution is shown inFIG. 8D. Each distribution represents measurements of >800 tricepsmuscle fibers from 7 animals (>100 measurements/animal); P<0.0001. Theeffect of ursolic acid on peak grip strength (normalized to body weight)is shown in FIG. 8E. Each data point represents one mouse, andhorizontal bars denote the means. Non-normalized grip strength data were157±9 g (control diet) and 181±6 g (ursolic acid diet) (P=0.04).

Moreover, dietary ursolic acid increased the specific force generated bymuscles ex vivo (FIG. 9). Briefly, six-week old male C57BL/6 mice wereprovided either standard diet or diet containing 0.27% ursolic acid for16 weeks before being euthanized. The lower hindlimb was removed (bytranssecting the upper hindlimb mid-way through the femur), and placedin Krebs solution aerated with 95% O2 and 5% CO2. The gastrocnemius,soleus and tibialis anterior muscles, as well as the distal half of thetibia and fibula were then removed and discarded, leaving the extensordigitorum longus and peroneus muscles with their origins and insertionsintact. A suture was placed through the proximal tendon and secured tothe distal femur fragment. This ex vivo preparation was then mountedvertically in a water jacket bath (Aurora Scientific 1200A Intact MuscleTest System, filled with aerated Krebs solution) by attaching the sutureto a servo-controlled lever (superiorly) and clamping the metatarsals(inferiorly). Passive muscle force was adjusted to a baseline of 1 g,and then muscles were stimulated with supramaximal voltage (80 V) at 100Hz. The mean time from euthanasia to maximal force measurements was 10min. After force measurements, muscles were removed and weighed in orderto calculate specific titanic force. Maximal tetanic force and muscleweight did not differ between the two groups (P=0.20 and 0.26,respectively). Data are means±SEM from 5-6 mice per diet. P-values weredetermined with a t-test. Together, the data in FIGS. 6 and 7 providemorphological and functional evidence that ursolic acid induced skeletalmuscle hypertrophy.

7. Ursolic Acid Induces Trophic Changes in Skeletal Muscle GeneExpression.

The foregoing results suggested that ursolic acid might alter skeletalmuscle gene expression. To test this hypothesis, an unbiased approachwas used, specifically exon expression arrays were used to analyzegastrocnemius muscle mRNA expression in mice that had been fed dietslacking or containing ursolic acid for 5 weeks. Mice were provided adlib access to either standard chow (control diet) or standard chowsupplemented with 0.27% ursolic acid (ursolic acid diet) for 5 weeksbefore gastrocnemius muscle RNA was harvested and analyzed by AffymetrixMouse Exon 1.0 ST arrays (n=4 arrays per diet). Each array assessedpooled gastrocnemius RNA from two mice. Stringent criteria were used forursolic acid-induced effects on mRNA levels (P<0.005), and mRNAs withlow levels of expression were disregarded (i.e. only transcripts thatwere increased to a mean log₂ hybridization signal≥8, or repressed froma mean log 2 hybridization signal≥8 were included). The results werethat ursolic acid decreased 18 mRNAs and increased 51 mRNAs (outof >16,000 mRNAs analyzed. The results are shown in Table X4 (“Change”is the meang log₂ change or difference between mice on ursolic acid dietand control diet, i.e. [Mean log₂ mRNA levels in ursolic acid diet]minus [Mean log₂ mRNA levels in control diet]).

TABLE X4 Mouse skeletal muscle mRNAs induced or repressed by ursolicacid. mRNA Protein Change P Smox spermine oxidase 0.81 0.001 Lyz2lysozyme 2 0.71 0.001 C3 complement component 3 0.70 0.000 Tyrobp TYROprotein tyrosine 0.69 0.001 kinase binding protein Lum lumican 0.610.001 Igf1 insulin-like growth 0.56 0.005 factor 1 Fmo1 flavincontaining 0.47 0.000 monooxygenase 1 Ostn osteocrin 0.43 0.001 Namptnicotinamide 0.41 0.003 phosphoribosyltransferase H19 H19 fetal livermRNA 0.39 0.004 Hipk2 homeodomain interacting 0.38 0.002 protein kinase2 Fbp2 fructose bisphosphatase 2 0.37 0.003 Gpx1 glutathione peroxidase1 0.36 0.001 Sepp1 selenoprotein P, plasma, 1 0.35 0.004 Parp3 poly(ADP-ribose) 0.32 0.001 polymerase family, member 3 Hspb8 heat shockprotein 8 0.32 0.000 Musk muscle, skeletal, 0.31 0.004 receptor tyrosinekinase Fhl3 four and a half LIM 0.31 0.005 domains 3 Hsph1 heat shock105 kDa/110 0.30 0.001 kDa protein 1 Arfgap2 ADP-ribosylation factor0.30 0.001 GTPase activating protein 2 Cd24a CD24a antigen 0.28 0.002Sepx1 selenoprotein X 1 0.28 0.003 Hk2 hexokinase 2 0.26 0.003 Ggctgamma-glutamyl 0.24 0.005 cyclotransferase Trip10 thyroid hormonereceptor 0.23 0.000 interactor 10 Npc1 Niemann Pick type C1 0.22 0.001Asb5 ankyrin repeat and SOCs 0.21 0.001 box-containing 5 Vps29 vacuolarprotein sorting 0.20 0.000 29 (S. pombe) Ahsa2 AHA1, activator of heat0.18 0.001 shock protein ATPase homolog 2 Lsm14a LSM14 homolog A (SCD6,0.18 0.004 S. cerevisiae) Pdha1 pyruvate dehydrogenase 0.18 0.001 E1alpha 1 Trappc2l trafficking protein 0.16 0.004 particle complex 2-likeUbe2l3 ubiquitin-conjugating 0.16 0.003 enzyme E2L 3 Ctsb cathepsin B0.16 0.003 D0H4S114 DNA segment, human 0.15 0.004 D4S114 Psma2proteasome (prosome, 0.15 0.005 macropain) subunit, alpha type 2 Mrpl46mitochondrial ribosomal 0.15 0.001 protein L46 Eef1e1 eukaryotictranslation 0.15 0.002 elongation factor 1 epsilon 1 Krr1 KRR1, smallsubunit 0.15 0.005 (SSU) processome component, homolog Ndufaf4 NADHdehydrogenase 0.14 0.005 (ubiquinone) 1 alpha subcomplex, assemblyfactor 4 Ndufs2 NADH dehydrogenase 0.14 0.002 (ubiquinone) Fe—S protein2 2610507B11Rik RIKEN cDNA 2610507B11 gene 0.14 0.000 Ssr4 signalsequence receptor, delta 0.14 0.000 Ndufs4 NADH dehydrogenase 0.14 0.003(ubiquinone) Fe—S protein 4 Sqstm1 sequestosome 1 0.12 0.001 Gfm1 Gelongation factor, 0.12 0.003 mitochondrial 1 2310016M24Rik RIKEN cDNA2310016M24 gene 0.12 0.004 Sod2 superoxide dismutase 2, 0.12 0.001mitochondrial Prdx5 peroxiredoxin 5 0.10 0.005 BC004004 cDNA sequenceBC004004 0.06 0.001 Ghitm growth hormone inducible 0.05 0.005transmembrane protein Foxn3 forkhead box N3 −0.09 0.000 Klhl31kelch-like 31 (Drosophila) −0.09 0.001 Acadm acyl-Coenzyme Adehydrogenase, −0.11 0.001 medium chain Eif4g3 eukaryotic translationinitiation −0.12 0.005 factor 4 gamma, 3 Nrap nebulin-related anchoringprotein −0.14 0.003 Golga4 golgi autoantigen, golgin subfamily −0.140.003 a, 4 Paip2b poly(A) binding protein −0.16 0.000 interactingprotein 2B Pde4dip phosphodiesterase 4D interacting −0.18 0.001 protein(myomegalin) Sfpq splicing factor proline/glutamine −0.18 0.005 rich Pnnpinin −0.18 0.002 D4Wsu53e DNA segment, Chr 4, Wayne −0.18 0.003 StateUniversity 53, expressed Mlec malectin −0.19 0.003 Cacna1s calciumchannel, voltage- −0.22 0.001 dependent, L type, alpha 1S Sfrs5 splicingfactor, arginine/serine- −0.22 0.005 rich 5 (SRp40, HRS) Nntnicotinamide nucleotide −0.24 0.002 transhydrogenase Adprhl1ADP-ribosylhydrolase like 1 −0.26 0.002 Ddit4l DNA-damage-inducibletranscript −0.32 0.000 4-like Fbxo32 F-box protein 32 (Atrogin-I) −0.350.001

As discussed above, atrogin-1 and MuRF1 are transcriptionallyup-regulated by atrophy-inducing stresses (see FIG. 4B and Sacheck J M,et al. (2007) Faseb J 21(1): 140-155), and they are required for muscleatrophy (Bodine S C, et al. (2001) Science (New York, N.Y. 294(5547):1704-1708). Moreover, in the studies of fasted mice as described hereinabove, ursolic acid reduced atrogin-1 and MuRF1 mRNAs (FIG. 6H).Consistent with that finding, the arrays indicated that dietary ursolicacid reduced atrogin-1 mRNA, which was the most highly repressed mRNA(FIG. 10A). The results shown in FIG. 10A represent a subset of themRNAs from Table X4 which had the greatest increase or decrease inexpression level in response to ursolic acid. Although MuRF1 mRNA wasnot measured by the arrays used in these experiments, qPCR analysisconfirmed that dietary ursolic acid repressed both atrogin-1 and MuRF1mRNAs (FIG. 10B; data are means±SEM). Interestingly, one of the mosthighly up-regulated muscle mRNAs was IGF1 (FIGS. 8A and 8B), whichencodes insulin-like growth factor-I (IGF-I), a locally generatedautocrine/paracrine hormone. IGF1 mRNA is known to be transcriptionallyinduced in hypertrophic muscle (Hameed M, et al. (2004) The Journal ofphysiology 555 (Pt 1):231-240; Adams G R & Haddad F (1996) J ApplPhysiol 81(6):2509-2516; Gentile M A, et al. (2010) Journal of molecularendocrinology 44(1):55-73). In addition, increased skeletal muscle IGF1expression reduces denervation-induced muscle atrophy (Shavlakadze T, etal. (2005) Neuromuscul Disord 15(2): 139-146), and stimulates musclehypertrophy (Barton-Davis E R, et al. (1998) Proceedings of the NationalAcademy of Sciences of the United States of America 95(26): 15603-15607;Musarò A, et al. (2001) Nature Genetics 27(2):195-200). Moreover, bystimulating skeletal muscle insulin/IGF-I signaling, IGF-I repressesatrogin-1 and MuRF mRNAs (Sacheck J M, et al. (2004) Am J PhysiolEndocrinol Metab 287(4):E591-601; Frost R A, et al. (2009) J CellBiochem 108(5): 1192-1202), as well as DDIT4L mRNA (ibid), which, afteratrogin-1 mRNA, was the second most highly repressed mRNA in muscle fromursolic acid-treated mice (FIG. 10A). Thus, 5 weeks of dietary ursolicacid altered skeletal muscle gene expression in a manner known to reduceatrophy and promote hypertrophy, and muscle-specific IGF1 inductionemerged as a likely contributing mechanism in ursolic acid-inducedmuscle hypertrophy. The effect of ursolic acid on plasma IGF-I levelswas also determined, which primarily reflect growth hormone-mediatedhepatic IGF-I production (Yakar S, et al. (1999) Proceedings of theNational Academy of Sciences of the United States of America96(13):7324-7329). Although diets containing 0.14% or 0.27% ursolic acidincreased muscle mass (described in greater detail below; FIG. 12A),neither increased plasma IGF-I (FIG. 10C). For the data in FIG. 10C,mice were provided ad lib access to either standard chow (control diet)or standard chow supplemented with the indicated concentration ofursolic acid for 7 weeks before plasma IGF-I levels were measured. Eachdata point represents one mouse, and horizontal bars denote the means.P-values were determined by one-way ANOVA with Dunnett's post-test. Ofnote, exon expression arrays indicated that ursolic acid increasedlevels of all measured IGF1 exons (exons 2-6; FIG. 11A). The data inFIG. 11A are mean exon-specific log₂ hybridization signals from thearrays described in Table X2. However, ursolic acid did not alter levelsof mRNAs encoding myostatin (which reduces muscle mass, for example seeLee S J (2004) Annu Rev Cell Dev Biol 20:61-86), or twist or myogenin(which are induced by IGF-I during development, for example see DupontJ, et al. (2001) The Journal of biological chemistry276(28):26699-26707; Tureckova J, et al. (2001) The Journal ofbiological chemistry 276(42):39264-39270). Moreover, ursolic acid didnot alter the amount of IGF1 mRNA in adipose tissue (FIG. 11B). Briefly,the data shown in FIG. 11B were obtained as follows: mice were providedad lib access to either standard chow (control diet) or standard chowsupplemented with 0.27% ursolic acid (ursolic acid diet) for 7 weeksbefore retroperitoneal adipose tissue was harvested for qPCRquantification of IGF1 mRNA. The data shown are means±SEM from 5 miceper group. Without wishing to be bound by a particular theory, ursolicacid-mediated IGF1 induction may be localized to skeletal muscle.

8. Ursolic Acid Enhances Skeletal Muscle IGF-I Signaling.

Although muscle-specific IGF1 induction is characteristic of, andcontributes to, muscle hypertrophy, it may be a relatively late eventthat promotes hypertrophy after it has been initiated by other stimuli(Adams G R, et al. (1999) J Appl Physiol 87(5):1705-1712). Withoutwishing to be bound by a particular theory, it is possible that ursolicacid might have a more proximal effect on insulin/IGF-I signaling. In aprevious study of non-muscle cell lines (CHO/IR and 3T3-L1 cells),ursolic acid enhanced insulin-mediated Akt activation (Jung S H, et al.(2007) The Biochemical journal 403(2):243-250). To determine whetherursolic acid might have a similar effect in skeletal muscle, the levelof phosphorylated Akt was assessed in quadriceps muscles of mice feddiets lacking or containing ursolic acid. Briefly, mice were provided adlib access to either standard chow (control diet) or standard chowsupplemented with 0.27% ursolic acid for 16 weeks. Total proteinextracts from quadriceps muscles were subjected to SDS-PAGE, followed byimmunoblot analysis for phosphorylated and total Akt, as indicated. Arepresentative immunoblot is shown in FIG. 10D. Immunoblot data werequantitated as follows: in each mouse, the level of phospho-Akt wasnormalized to the level of total Akt; these ratios were then normalizedto the average phospho-Akt/total Akt ratio from control mice and theresults are shown in FIG. 10E (data are means±SEM from 9 mice per diet.P-value was determined by unpaired t-test). The data show that inquadriceps, ursolic acid increased Akt phosphorylation by 1.8-fold.

The effect of ursolic acid on Akt activation was examined in C2C12skeletal myotubes, a well-established in vitro model of skeletal muscle(Sandri M, et al. (2004) Cell 117(3):399-412; Stitt T N, et al. (2004)Mol Cell 14(3):395-403). Use of an in vitro system, such as C2C12skeletal myotubes, circumvented potentially confounding effects fromnon-muscle tissues, and enabled a determination of whether IGF-I orinsulin was required for ursolic acid's effect. The latter considerationwas important because circulating IGF-I and insulin are always presentin healthy animals. Use of an in vitro system also allowed testing of aclearly defined concentration of ursolic acid (10 μM, similar what wasused in the Connectivity Map (8.8 μM)) for a clearly defined time ofincubation (20 min). These considerations were important because the invivo pharmacokinetic properties of ursolic acid are not yet known.

For the data shown in FIGS. 8F-8K, serum-starved C2C12 myotubes weretreated in the absence or presence of ursolic acid (10 μM) and/or IGF-I(10 nM), as indicated. For studies of the IGF-I receptor, cells wereharvested 2 min later, and protein extracts were subjected toimmunoprecipitation with anti-IGF-I receptor β antibody, followed byimmunoblot analysis with anti-phospho-tyrosine or anti-IGF-I receptor βantibodies to assess phospho- and total IGF-I receptor, respectively.For other studies, cells were harvested 20 min after addition of ursolicacid and/or IGF-I, and immunoblot analyses were performed using totalcellular protein extracts and antibodies specific for the phosphorylatedor total proteins indicated. Representative immunoblots showing effectof ursolic acid on IGF-I-mediated phosphorylation of Akt (FIG. 10F), S6K(FIG. 10G) and IGF-I receptor (FIG. 10H). Data from immunoblots wasquantitated as follows: levels in the presence of ursolic acid and IGF-Iwere normalized to levels in the presence of IGF-I alone, which were setat 1 and are indicated by the dashed line. The data shown in FIG. 10Iare means±SEM from ≥3 experiments.

For the data shown in FIGS. 9C-9F, serum-starved C2C12 myotubes weretreated in the absence or presence of ursolic acid (10 μM), insulin (10nM) and/or IGF-I (10 nM), as indicated. For studies of the insulinreceptor, cells were harvested 2 min later, and protein extracts weresubjected to immunoprecipitation with anti-insulin receptor β antibody,followed by immunoblot analysis with anti-phospho-insulin receptor β(Y1162/1163) or anti-insulin receptor β antibodies to assess phospho-and total insulin receptor, respectively. For other studies, cells wereharvested 20 min after addition of ursolic acid, insulin and/or IGF-I,and immunoblot analyses were performed using total cellular proteinextracts and antibodies specific for the phosphorylated or totalproteins indicated.

When serum-starved myotubes were treated with ursolic acid alone, Aktphosphorylation did not increase (FIG. 10F). However, in the presence ofIGF-I, ursolic acid increased Akt phosphorylation by 1.9-fold (FIGS. 8Fand 8I). Ursolic acid also increased Akt phosphorylation in the presenceof insulin (FIG. 11C). Thus, ursolic acid enhanced IGF-I-mediated andinsulin-mediated Akt phosphorylation. The finding that ursolic acidenhanced muscle Akt activity in vivo and in vitro was consistent withthe finding that ursolic acid's mRNA expression signature negativelycorrelated with the mRNA expression signatures of LY-294002 andwortmannin (FIGS. 4B and 5B), which inhibit insulin/IGF-I signalingupstream of Akt. However, ursolic acid's signature also negativelycorrelated with the signature of rapamycin, which inhibits insulin/IGF-Isignaling downstream of Akt.

Although ursolic acid alone did not increase S6K phosphorylation (FIG.11D), it enhanced IGF-I-mediated and insulin-mediated S6Kphosphorylation (FIGS. 8G, 8I and 9D). To further investigate themechanism, the effect of ursolic acid on the IGF-I receptor wasexamined. Ursolic acid increased IGF-I receptor phsophorylation in thepresence but not the absence of IGF-I (FIGS. 8H and 8I). Similarly,ursolic acid increased insulin receptor phosphorylation in the presencebut not the absence of insulin (FIG. 11E). Both of these effects wererapid, occurring within 2 minutes after the addition of ursolic acid andeither IGF-I or insulin. Consistent with enhanced signaling at the levelof the IGF-I and insulin receptors, ursolic acid also enhancedIGF-I-mediated and insulin-mediated ERK phosphorylation (FIGS. 8J and9F). Moreover, ursolic acid enhanced IGF-I-mediated phosphorylation(inhibition) of FoxO transcription factors, which activate transcriptionof atrogin-1 and MuRF1 mRNAs (FIG. 10K; Sandri M, et al. (2004) Cell117(3):399-412; Stitt T N, et al. (2004) Mol Cell 14(3):395-403.).Without wishing to be bound by a particular theory, ursolic acidrepresses atrophy-associated gene expression and promotes musclehypertrophy by increasing activity of the IGF-I and insulin receptors.

9. Ursolic Acid Reduces Adiposity.

Mice were provided ad lib access to standard chow supplemented with theindicated concentration (weight percent in chow, either 0.14% or 0.28%as indicated in FIG. 12) of ursolic acid for 7 weeks before tissues wereharvested for analysis. Data are means±SEM from 10 mice per diet. Datafor the effects of ursolic acid on weights of skeletal muscle(quadriceps+triceps), epididymal fat, retroperitoneal fat and heart areshown in FIG. 12A. The P-values, determined by one-way ANOVA withpost-test for linear trend, were <0.001 for muscle; 0.01 and 0.04 forepididymal and retroperitoneal fat, respectively; and 0.46 for heart.The data show that 7 weeks of dietary ursolic acid increased skeletalmuscle weight in a dose-dependent manner, with a peak effect at 0.14%ursolic acid. Interestingly, although ursolic acid increased muscleweight, it did not increase total body weight (FIG. 12B; P-values were0.71 and 0.80 for initial and final weights, respectively).

The data in FIG. 12A also show that 7 weeks of dietary ursolic acidreduced the weight of epididymal and retroperitoneal fat depots, with apeak effect at 0.14%. In another study, mice were provided ad lib accessto either standard chow (control diet) or standard chow supplementedwith 0.27% ursolic acid (ursolic acid diet) for 5 weeks. Therelationship between skeletal muscle weight (quadriceps, triceps,biceps, TA, gastrocnemius and soleus) and retroperitoneal adipose weightis shown in FIG. 12C. Each data point in FIG. 12C represents one mouse;P<0.001 for both muscle and adipose by unpaired t-test. The data showthat 5 weeks of ursolic acid administration (0.14%) also reduced adiposeweight. Thus, muscle and fat weights were inversely related. Withoutwishing to be bound by a particular theory, ursolic acid-treated micecontain less fat because, in part, ursolic acid increases Akt activity(see FIGS. 8 and 9), and muscle-specific increases in Akt activityreduce adiposity as a secondary consequence of muscle hypertrophy (Lai KM, et al. (2004) Molecular and cellular biology 24(21):9295-9304;Izumiya Y, et al. (2008) Cell metabolism 7(2): 159-172).

Ursolic acid reduced adipose weight by reducing adipocyte size as shownby data in FIGS. 10D-10F. FIG. 12D shows a representative H&E stain ofretroperitoneal fat for animals feed a control data or a chow with 0.27%ursolic acid as indicated. The data in FIG. 12D are shown quantitativelyin FIG. 12E in terms of adipocyte diameter, where data point representsthe average diameter of ≥125 retroperitoneal adipocytes from one mouse.The retroperitoneal adipocyte size distribution. Each distributionrepresents combined adipocyte measurements (>1000 per diet) from FIG.12E.

The changes in adipocyte size were accompanied by a significantreduction in plasma leptin levels, which correlated closely with adiposeweight (see FIGS. 10G and 10H). In FIG. 12G, each data point representsone mouse, and horizontal bars denote the means. P-values weredetermined by t-test. In FIG. 12H, each data point represents one mouse.Importantly, ursolic acid also significantly reduced plasma triglyceride(FIG. 12I) and cholesterol (FIG. 12J). In FIGS. 10I and 10J, each datapoint represents one mouse, and horizontal bars denote the means.P-values were determined by unpaired t-test. Although ursolic acidreduced leptin, it did not alter food intake (FIG. 13A). In this study,mice were provided ad lib access to either standard chow (control diet)or standard chow supplemented with 0.27% ursolic acid (ursolic aciddiet) for 4 weeks. Mice were then moved to a comprehensive animalmetabolic monitoring system (CLAMS; Columbus Instruments, Columbus,Ohio) and provided with ad lib access to the same diets. Foodconsumption was measured for 48 hours. Data are means±SEM from 6 miceper group. However, ursolic acid did not alter weights of heart (FIG.12A), liver or kidney (FIGS. 11B and 11C), nor did it elevate plasmamarkers of hepatotoxicity or nephrotoxicity (alanine aminotransferase,bilirubin and creatinine; see FIGS. 11D-11F). The data in FIGS. 11B-11Fwere obtained as follows: mice were provided ad lib access to eitherstandard chow (control diet) or standard chow supplemented with 0.27%ursolic acid (ursolic acid diet) for 5 weeks before tissues and plasmawere harvested for the indicated measurements; each data pointrepresents one mouse, and horizontal bars denote the means. For FIG. 13,P-values were determined with unpaired t-tests. Thus, dietary ursolicacid had two major effects: skeletal muscle hypertrophy and reducedadiposity.

10. Ursolic Acid Reduces Weight Gain and White Adipose Tissue.

The findings that ursolic acid increased skeletal muscle and decreasedadiposity suggested that ursolic acid might increase energy expenditure,which would lead to obesity resistance. To test this, C57BL/6 mice weregiven ad libitum access to a high fat diet (HFD; Teklad TD.93075; 55%calories from fat) lacking or containing 0.27% ursolic acid. After 7weeks, mice from each group were studied for three days in comprehensivelab animal monitoring systems (“CLAMS”; Columbus Instruments). In theCLAMS, mice were maintained on the same diet they had been eating sincethe beginning of the experiment. Following CLAMS, tissues were harvestedfor analysis. In high fat-fed mice, ursolic acid dramatically reducedweight gain, and this effect was apparent within one week (FIG. 14A). Aspreviously observed in mice fed ursolic acid and standard chow (FIG. 8),ursolic acid increased grip strength and muscle mass (FIGS. 12B and12C). Moreover, ursolic acid reduced retroperitoneal and epididymal fat(FIGS. 12D and 12E). Interestingly, in the scapular fat pad, whichcontains a mixture of white and thermogenic brown fat, ursolic acidreduced white fat (FIG. 14F), but increased brown fat (FIG. 14G).Importantly, increased skeletal muscle and brown adipose tissue would bepredicted to increase energy expenditure. Indeed, CLAMS revealed thatursolic acid increased energy expenditure (FIG. 14H), providing anexplanation for how ursolic acid reduces adiposity and obesity.Remarkably, CLAMS analysis revealed that ursolic acid-treated miceconsumed more food (FIG. 14I), even though they gained less weight (FIG.14A). For the data shown in FIG. 14A, data are means±SEM from 12 controlmice and 15 treated mice, but it should be noted that some error barsare too small to see; P<0.01 at 1 wk and each subsequent time point. InFIGS. 12B-12I, each data point represents one mouse and horizontal barsdenote the means. P-values were determined with unpaired t-tests.

11. Ursolic Acid Reduces Obesity-Related Pre-Diabetes, Diabetes, FattyLiver Disease and Hypercholesterolemia.

The study was carried out as follows: C57BL/6 mice were given ad libitumaccess to a high fat diet (“HFD”; Teklad TD.93075; 55% calories fromfat) lacking or containing 0.27% ursolic acid. After 5 weeks, mice werefasted for 16 h before blood glucose was measured via the tail vein(FIG. 15A). Normal fasting blood glucose: ≤100 mg/dl. (B-I) After 7weeks, liver and plasma were harvested for analysis (FIGS. 13B-13I). Thedata shown in FIG. 15A suggest that most mice fed HFD without ursolicacid for 6 weeks developed impaired fasting glucose (pre-diabetes) ordiabetes. Importantly, this was prevented by ursolic acid (FIG. 15A). Inaddition, mice fed HFD without ursolic acid developed fatty liverdisease, as evidenced by increased liver weight (>30% increase abovenormal mouse liver weight of 1500 mg; FIG. 15B), hepatocellular lipidaccumulation (FIG. 15C, H&E stain at 20× magnification; FIG. 15D,lipid-staining osmium at 10× magnification), and elevated plasma liverfunction tests (FIG. 15E, AST; 13F, ALT; 13G, alkaline phosphatase(labeled as “Alk. Phos. in figure); and, 13H, cholesterol). However,ursolic acid prevented all of these hepatic changes (FIG. 15B-13G). Inaddition, ursolic acid reduced obesity-related hypercholesterolemia(FIG. 15H). In FIGS. 13A, 13B, and 13E-13H, each data point representsone mouse and horizontal bars denote the means.

12. Oleanolic Acid does not Increase Skeletal Muscle Mass.

The effect of ursolic acid on skeletal muscle weight and liver weightwas compared to the effects by oleanolic acid and metformin. Metforminwas a compound identified from muscle atrophy signature-1, but notmuscle atrophy signature-2. Oleanolic acid, like ursolic acid is apentacyclic acid triterpane. This is a structurally similar compound toursolic acid. However, the two compounds are distinct: oleanolic acidhas two methyl groups at position 20, whereas ursolic acid has a singlemethyl group at each of positions 19 and 20 (compare FIGS. 14A and 14D).Both ursolic acid and oleanolic acid reduce blood glucose, adiposity andhepatic steatosis (Wang Z H, et al. (2010) European journal ofpharmacology 628(1-3):255-260; Jayaprakasam B, et al. (2006) J AgricFood Chem 54(1):243-248; de Melo C L, et al. (2010) Chem Biol Interact185(1):59-65). In addition, both ursolic acid and oleanolic acid possessa large number of cellular effects and biochemical targets, includingnearly equivalent inhibition of protein tyrosine phosphatases (“PTPs”;see Zhang W, et al. (2006) Biochimica et biophysica acta1760(10):1505-1512; Qian S, et al. (2010) J Nat Prod 73(11):1743-1750;Zhang Y N, et al. (2008) Bioorg Med Chem 16(18):8697-8705). However, theeffects of these compounds on skeletal muscle mass were not known.

Because some PTPs (particularly PTP1B) dephosphorylate (inactivate) theinsulin receptor, PTP inhibition represented a potential mechanism toexplain ursolic acid-mediated enhancement of insulin signaling. Thus,because oleanolic acid and ursolic acid inhibit PTP1B and other PTPswith similar efficacy and potency in vitro (Qian S, et al. (2010) J NatProd 73(11):1743-1750; Zhang Y N, et al. (2008) Bioorg Med Chem16(18):8697-8705), testing oleanolic acid's effects on skeletal masstests the potential role of PTP inhibition. It should be noted thatneither ursolic acid nor oleanolic acid is known to inhibit PTPs invivo, and neither of these compounds are known to enhance IGF-Isignaling. Moreover, ursolic acid's capacity to inhibit PTPs has beendisputed based on ursolic acid's failure to delay insulin receptorde-phosphorylation in cultured cells (Jung S H, et al. (2007) TheBiochemical journal 403(2):243-250), and ursolic acid's capacity to actas an insulin mimetic (Jung S H, et al. (2007) The Biochemical journal403(2):243-250). In addition, global and muscle-specific PTP1B knockoutmice do not possess increased muscle mass, although they are resistantto obesity and obesity-related disorders (Delibegovic M, et al. (2007)Molecular and cellular biology 27(21):7727-7734; Klaman L D, et al.(2000) Molecular and cellular biology 20(15):5479-5489). Furthermore,ursolic acid increases pancreatic beta cell mass and serum insulinlevels in vivo, perhaps via its anti-inflammatory effects (Wang Z H, etal. (2010) European journal of pharmacology 628(1-3):255-260;Jayaprakasam B, et al. (2006) J Agric Food Chem 54(1):243-248; de Melo CL, et al. (2010) Chem Biol Interact 185(1):59-65). Importantly,inflammation is now recognized as a central pathogenic mechanism inmuscle atrophy, metabolic syndrome, obesity, fatty liver disease andtype 2 diabetes. Thus, the existing data suggest at least fourmechanisms to explain ursolic acid's capacity to increase insulinsignaling in vivo: PTP inhibition, direct stimulation of the insulinreceptor, increased insulin production, and reduced inflammation. Ofthese four potential mechanisms, only the latter three have beendemonstrated in vivo.

To compare the effects of ursolic acid and oleanolic acid on skeletalmuscle and liver weight, C57BL/6 mice were administered ursolic acid(200 mg/kg), oleanolic acid (200 mg/kg), or vehicle alone (corn oil) viai.p. injection. Mice were then fasted, and after 12 hours of fasting,mice received a second dose of ursolic acid, oleanolic acid, or vehicle.After 24 hours of fasting, lower hindlimb skeletal muscles and liverwere harvested and weighed. As shown previously, ursolic acid increasedskeletal muscle weight (FIG. 16B), but not liver weight (FIG. 16C). Incontrast, oleanolic acid increased liver weight (FIG. 14F), but notskeletal muscle weight (FIG. 16E). Interestingly, metformin (250 mg/kg)resembled oleanolic acid in biological effect: it increased liver weight(FIG. 16I), but not muscle weight (FIG. 16H). Without wishing to bebound by a particular theory, ursolic acid increases skeletal muscle andinhibit muscle atrophy by a pathway that does not involve PTPinhibition.

13. Targeted Inhibition of PTP1B does not Induce Skeletal MuscleHypertrophy.

To further rule out the potential role of PTP1B inhibition in skeletalmuscle hypertrophy, PTP1B expression was specifically reduced in mouseskeletal muscle by transfecting plasmid DNA constructed to express RNAinterference constructs. Briefly, C57BL/6 mouse tibialis anteriormuscles were transfected with 20 μg pCMV-miR-control (control plasmidtransfected in the left TA) or either 20 μg pCMV-miR-PTP1B #1 (encodingmiR-PTP1B #1; transfected in the right TA) or 20 μg pCMV-miR-PTP1B #2(encoding miR-PTP1B #2; transfected in the right TA). miR-PTP1B #1 andmiR-PTP1B #2 encode two distinct RNA interference (RNAi) constructstargeting distinct regions of PTP1B mRNA. Tissue was harvested 10 daysfollowing transfection.

Of note with regard to FIG. 17A, mRNA measurements were taken from theentire TA muscle. Because electroporation transfects only a portion ofmuscle fibers, the data underestimate PTP1B knockdown in transfectedmuscle fibers. In FIG. 17A, mRNA levels in the right TA were normalizedto levels in the left TA, which were set at 1; data are means±SEM from 3mice. In FIG. 17B, in each TA muscle, the mean diameter of >300transfected fibers was determined; data are means±SEM from 3 TA musclesper condition. For both FIGS. 15A and 15B, P-values were determined withone-tailed paired t-tests.

Although both miR-PTP1B constructs reduced PTP1B mRNA (FIG. 17A),neither increased skeletal muscle fiber diameter (FIG. 17B). These datademonstrate that targeted PTP1B inhibition does not cause muscle fiberhypertrophy. Without wishing to be bound by a particular theory, ursolicacid does not increase skeletal muscle by inhibiting PTP1B.

14. Ursolic Acid Serum Levels Associated with Increased Muscle Mass andDecreased Adiposity.

To determine the dose-response relationship between dietary ursolic acidand muscle and adipose weight, C57BL/6 mice were fed standard chowcontaining varying amounts of ursolic acid for 7 weeks. Serum ursolicacid levels from mice were determined as described above. As shownpreviously in FIG. 12A, ursolic acid increased skeletal muscle weightand decreased weight of retroperitoneal and epididymal fat pads in adose-dependent manner, but did not alter heart weight (FIG. 18A; dataare means±SEM). These effects of ursolic acid were discernable at 0.035%ursolic acid and were maximal at doses≥0.14% ursolic acid. Serum wascollected from these same mice at the time of necropsy, and thenmeasured random serum ursolic acid levels via ultra high performanceliquid chromatography (UPLC). The data indicate that ursolic acid serumlevels in the range of 0.25-0.5 μg/ml are sufficient to increase musclemass and decrease adiposity (FIG. 18B; data are means±SEM). Of note, 0.5μg/ml equals 1.1 μM ursolic acid, close to the dose used in theConnectivity Map (8.8 μM) and in the C2C12 experiments (10 μM) describedabove.

The data described herein indicate that ursolic acid reduced muscleatrophy and stimulated muscle hypertrophy in mice. Importantly, ursolicacid's effects on muscle were accompanied by reductions in adiposity,fasting blood glucose and plasma leptin, cholesterol and triglycerides,as well as increases in the ratio of skeletal muscle to fat, the amountof brown fat, the ratio of brown fat to white fat, and increased energyexpenditure. Without wishing to be bound by a particular theory, ursolicacid reduced muscle atrophy and stimulated muscle hypertrophy byenhancing skeletal muscle IGF-I expression and IGF-I signaling, andinhibiting atrophy-associated skeletal muscle mRNA expression.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. It will be apparent to those skilled in the art thatvarious modifications and variations can be made in the presentinvention without departing from the scope or spirit of the invention.More specifically, certain agents which are both chemically andphysiologically related can be substituted for the agents describedherein while the same or similar results can be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims. Other embodiments of theinvention will be apparent to those skilled in the art fromconsideration of the specification and practice of the inventiondisclosed herein. It is intended that the specification and examples beconsidered as exemplary only, with a true scope and spirit of theinvention being indicated by the following claims.

15. Treatment of Muscle Atrophy

Several compounds have been shown to treat muscle atrophy as shownbelow.

a. Betulinic Acid

Betulinic acid has the following structure:

The mRNA expression signature of betulinic acid negatively correlated tohuman muscle atrophy signature-2. Therefore betulinic acid, like ursolicacid, could inhibit skeletal muscle atrophy. To test this, a mouse modelof immobilization-induced skeletal muscle atrophy was used: mice wereadministered vehicle (corn oil) or varying doses of ursolic acid(positive control) or betulinic acid via intraperitoneal injection twicea day for two days. One tibialis anterior (TA) muscle was immobilizedwith a surgical staple, leaving the contralateral mobile TA as anintra-subject control. The vehicle or the same dose of ursolic acid orbetulinic acid was continuously administered via i.p. injection twicedaily for six days before comparing weights of the immobile and mobileTAs. As expected, immobilization caused muscle atrophy, and ursolic acidreduced muscle atrophy in a dose-dependent manner, with maximalinhibition at 200 mg/kg (FIG. 19A). Betulinic acid also reduced muscleatrophy in a dose-dependent manner, with maximal inhibition at ≤50 mg/kg(FIG. 19B). These data indicate that betulinic acid reducesimmobilization-induced muscle atrophy, and it is more potent thanursolic acid.

b. Naringenin

Naringenin has the following structure:

The mRNA expression signature of naringenin negatively correlated tohuman muscle atrophy signatures-1 and -2. Therefore naringenin couldinhibit skeletal muscle atrophy. To test this, mice were administeredvehicle (corn oil), ursolic acid (200 mg/kg), naringenin (200 mg/kg), orthe combination of ursolic acid and naringenin (each at 200 mg/kg) viai.p injection twice a day for two days. One tibialis anterior (TA)muscle was immobilized with a surgical staple, leaving the contralateralmobile TA as an intrasubject control. Vehicle or the same doses ofursolic acid and/or naringenin was continuously administered via i.p.injection twice daily for six days before comparing weights of theimmobile and mobile TAs. Like ursolic acid, naringenin reduced muscleatrophy (FIG. 20). The combination of ursolic acid and naringenin alsoreduced muscle atrophy, but not more than either compound alone (FIG.20). These data indicate that naringenin reduces skeletal muscleatrophy.

Like ursolic acid, naringenin reduces blood glucose, as well as obesityand fatty liver disease. Therefore ursolic acid and naringenin couldhave additive effects. To determine this, weight-matched mice wereprovided ad libitum access to standard (Harlan Teklad formula 7013),high fat diet (HFD; Harlan Teklad formula TD93075), or HFD containingvarying concentrations of ursolic acid (0.15%) and/or naringenin (0.5%or 1.5%). After the mice consumed these diets for 5 weeks, fasting bloodglucose, total body weight, fat mass, liver weight, grip strength, andskeletal muscle weight was measured. As expected, HFD increased bloodglucose, and this increase in blood glucose was partially prevented byursolic acid and naringenin (FIG. 21A). The combination of ursolic acidplus either dose of naringenin reduced blood glucose more than eithercompound alone, and it restored blood glucose to normal levels (FIG.21A). Importantly, ursolic acid and naringenin did not have additiveeffects on total body weight (FIG. 21B), fat mass (FIG. 21C), liverweight (FIG. 21D), grip strength (FIG. 21E), or skeletal muscle weight(FIG. 21F). In addition, ursolic acid increased strength to a greaterextent than naringenin (FIG. 21E), and ursolic acid, but not naringenin,increased muscle weight (FIG. 21F). These differences between ursolicacid and naringenin in high fat fed mice indicates that ursolic acid andnaringenin have differences in their mechanisms of action, which couldexplain their additive effects on fasting blood glucose.

c. Tomatidine

Tomatidine has the following structure:

The mRNA expression signature of tomatidine negatively correlated tohuman muscle atrophy signatures-1 and -2. Therefore tomatidine couldinhibit skeletal muscle atrophy. To test this, mice were administeredvehicle (corn oil) or tomatidine (50, 100 or 200 mg/kg) via i.pinjection twice a day for two days. One tibialis anterior (TA) musclewas immobilized with a surgical staple, leaving the contralateral mobileTA as an intrasubject control. Vehicle or the same doses of tomatidinewas administered via i.p. injection twice daily for six days beforecomparing weights of the immobile and mobile TAs. All 3 doses oftomatidine reduced muscle atrophy, and the effect was maximal at 50mg/kg (FIG. 22A). The same protocol was used to compare the effects ofvehicle (corn oil) and tomatidine (5, 15 or 50 mg/kg) onimmobilization-induced muscle atrophy. Tomatidine reduced muscle atrophyin dose-dependent manner, with maximal effect at 50 mg/kg and EC_(50<5)mg/kg (FIG. 22B). These data indicate that tomatidine reducesimmobilization-induced muscle atrophy, and it is more potent thanursolic acid.

Tomatidine could also inhibit skeletal muscle atrophy induced byfasting. To test this, food was withdrawn from mice, and then vehicle,ursolic acid (200 mg/kg) or tomatidine (50 mg/kg) were administered byi.p. injection. Twelve hours later, mice received another i.p. injectionof vehicle or the same dose of ursolic acid or tomatidine. Twelve hourslater, skeletal muscles were harvested and weighed. Both ursolic acidand tomatidine increased skeletal muscle, indicating decreasedfasting-induced skeletal muscle atrophy (FIG. 23A). We next used thesame protocol to compare the effects of vehicle (corn oil) andtomatidine (5, 15 and 50 mg/kg). Tomatidine reduced muscle atrophy indose-dependent manner, with maximal effect at 50 mg/kg and EC₅₀ between5 and 15 mg/kg (FIG. 23B).

d. Allantoin, Tacrine, Ungerine, Hippeastrine and Conessine

Allantoin has the following structure:

Tacrine has the following structure:

Ungerine has the following structure:

Hippeastrine has the following structure:

Conessine has the following structure:

The mRNA expression signatures of allantoin, tacrine, ungerine(Prestwick-689), hippeastrine (Prestwick-675) and conessine alsonegatively correlated to human muscle atrophy signatures-1 and -2.Therefore these compounds could inhibit skeletal muscle atrophy. To testthis, the fasting-induced muscle atrophy model described above was usedto compare the effects of ursolic acid (200 mg/kg), tomatidine (50mg/kg), allantoin (2 mg/kg), tacrine (4 mg/kg), ungerine (2 mg/kg),hippeastrine (2 mg/kg) and conessine (2 mg/kg). Like ursolic acid andtomatidine, allantoin, tacrine, ungerine, hippeastrine and conessineincreased muscle weight in fasted mice (FIG. 24), indicating that thesecompounds decrease skeletal muscle atrophy.

Since ursolic acid and naringenin reduced fasting blood glucose,hippeastrine (2 mg/kg) and conessine (2 mg/kg) could have a similareffect. Hippeastrine and conessine reduced fasting blood glucose (FIG.25).

16. Prophetic Synthesis of Tacrine and Analogs

The formulas disclosed herein could be synthesized by reacting ananthranilonitrile derivative with a cyclohexanone derivative in thepresence of zinc chloride (Proctor et al., Curr Medici. Chem., 2000, 7,295-302). Such reaction is shown in Scheme 1A.

Thus, tacrine can be synthesized as shown in scheme 1B.

The formulas disclosed herein could also be synthesized by reacting anα-cyanocyclonones with a vide variety of anilines using either TiCl₄ orAlCl₃ as reagents (Proctor et al., Curr Medici. Chem., 2000, 7,295-302). An example of such reaction is shown in Scheme 1C.

Thus, tacrine could be synthesized as shown in scheme 1D.

17. Prophetic Synthesis of Naringenin and Analogs

The disclosed formulas could be synthesized as described in PCTapplication WO 2007/053915 by De Keukkeleire et al. which is herebyincorporated in its entirety by reference. In another example, Glucoylsubstituted naringenin could be extracted as described in U.S. Pat. No.6,770,630 by Kashiwaba et al. which is hereby incorporated in itsentirety by reference. As described by De Keukkeleire et al. thedisclosed formulas could be synthesized as shown in Scheme 2A:

The formation of the thioketone was described by Pathak, et al. (J. Org.Chem., 2008, 73, 2890-2893). The * in the scheme denotes moieties thatis or can be converted, using known chemistry, into the disclosed Rmoieties. For example, the synthesis of naringenin is shown in Scheme2B.

18. Prophetic Synthesis of Allantoin and Analogs

The disclosed formulas could be made using a variety of chemistry knownin the art. For example, one set of the disclosed formulas could be madeas shown in Scheme 3A and as described in U.S. Pat. No. 4,647,574 byIneaga et al, which is hereby incorporated herein by reference in itsentirety.

Allantoin could be prepared as described in U.S. Pat. No. 5,196,545 bySchermanz, which is hereby incorporated herein by reference in itsentirety, and as shown in Scheme 3B.

A comprehensive guide for how to make the disclosed formulas can befound in Kirk-Othmer Encyclopedia of Chemical Technology under thechapter Hydantoin and Its Derivatives by Avendaño et al (2000), which ishereby incorporated herein by reference in its entirety.

19. Prophetic Synthesis of Conessine and Analogs

Conessine is a steroid alkaloid found in plant species from theApocynaceae family, for example in Holarrhena floribunda. Conessinederivatives could be prepared as described in U.S. Pat. Nos. 3,539,449,3,466,279, and 3,485,825 by Marx, which are hereby incorporated byreference in their entirety. As described in U.S. Pat. Nos. 3,539,449,3,466,279, and 3,485,825 by Marx, conessine derivatives could beprepared using micro-organisms such as the fungus Stachybotrysparvispora and enzymes from Gloeosporium, Colletotrichum, andMyrothecium. For example, see Scheme 4A.

The conessine oxo derivatives could be further modified via a reductionand subsequent chemistry known to one skilled in the art, as shown inScheme 4B.

The hydroxyl functionality could undergo a number of chemical reactionsknown in the art. One example, as shown in Scheme 4B, is a Williamsonether synthesis.

Conessine derivatives could be prepared synthetically as described inU.S. Pat. No. 2,910,470, which is hereby incorporated by reference inits entirety. Conessine derivatives are also described in WO 2011/046978by Orlow, which is hereby incorporated by reference in its entirety.Synthesis of the disclosed formulas is also described in U.S. Pat. No.3,625,941 by Pappo, which is hereby incorporated in its entirety byreference.

20. Prophetic Synthesis of Tomatidine and Analogs

The formulas disclosed herein could be synthesized by the methoddisclosed by Uhle, and Moore, J. Am. Chem. Soc. 76, 6412 (1954); Uhle,J. Am. Chem. Soc. 83, 1460 (1961); and Kessar et al., Tetrahedron 27,2869 (1971), which are all hereby incorporated by reference in theirentirety. The disclosed compounds can also be made as shown in Scheme5A.

21. Prophetic Synthesis of Hippeastrine/Ungerine and Analogs

The disclosed formulas can be synthesized by method disclosed by Mañaset al. (J Am. Chem. Soc. 2010, 132, 5176-78), which is herebyincorporated by reference in its entirety. Thus, disclosed formulas canbe synthesized as shown in Scheme 6A.

Thus, for example, Hippeastrine can be made as shown in Scheme 6B.

Another route to make the disclosed formulas is shown in Scheme 6C, asdemonstrated by Mañas et al.

The disclosed derivatives can also be made using methods disclosed byHaning et al (Org. Biomolec. Chem. 2011, 9, 2809-2820).

22. Prophetic Synthesis of Betulinic Acid and Analogs

Betulininc acid analogs are also described in International Publishedapplication WO 2011/153315 by Regueiro-Ren et al. and in InternationalPublished application WO 2008/063318 by Safe et al. which are herebyincorporated by reference in its entirety. Betulinic acid analogs of thepresent invention of the present invention could be prepared genericallyas shown below in scheme 7A. The starting materials could be made withmethods known in the art.

Compounds are represented in generic form, with substituents as noted incompound descriptions elsewhere herein. A more specific example is setforth below in scheme 7B.

23. Muscle Atrophy Signature-3

Induced and repressed mRNA were evaluated for muscle atrophysignature-3. The statisitical significance for the identified mRNAs wasdefined as P≤0.01.

For induced mRNAs: mouse tibialis anterior mRNAs significantly inducedby 1 week of denervation and significantly induced by 24 h fasting.

For repressed mRNAs: mouse tibialis anterior mRNAs significantlyrepressed by 1 week of denervation and significantly repressed by 24 hfasting.

The identified induced mRNAs included 1200011I18Rik, 2310004I24Rik,Akap8l, Als2, Anapc7, Apod, Arrdc3, Atp6v1h, BC027231, Bsdc1, Ccdc77,Cd68, Cdkn1a, Ctps2, Ctsl, D930016D06Rik, Ddx21, Depdc7, Dido1, nttip2,Ece1, Eda2r, Egln3, Elk4, Erbb2ip, Errfi1, Fbxo30, Fbxo32, Fip1l1, Frg1,Gabarapl1, Gadd45a, Gnl2, Gnl3, Herpud2, Hpgd, Hspb7, Htatip2, Impact,Kdm3a, Klhl5, Lpin2, Med12, Mfap1b, Mgea5, Mknk2, Nmd3, Nup93, ORF19,Pacrgl, Parp4, Pdk4, Phc3, Plaa, Ppfibp1, Psma2, Ranbp10, Ranbp9,Rassf4, Riok1, Rlim, Sf3b1, Sik1, Slc20a1, Sin, Spag5, Srsf2ip, Syf2,Tbcld15, Tbk1, Tekt1, Tgif1, Tmem140, Tmem71, Tnks, Trim25, Trmt1,Tspyl2, Tsr1, Tulp3, Txlng, Ubfd1, Ubxn4, Utp14a, Wdr3, and Xpo4.

The identified repressed mRNAs included 1600014C10Rik, 1700021F05Rik,2310003L22Rik, 2310010M20Rik, 2310028O11Rik, 2310061C15Rik,2610528E23Rik, 2810432L12Rik, Abcd2, Acvr1, Aimp2, Ank1, Aqp4, Arl3,Asb10, Aurka, Bhlhe41, Bpnt1, Camk2a, Cby1, Cc2d2a, Cdc14a, Cdc42ep2,Clcn1, Cntfr, Col15a1, Col6a3, Cox11, Cox7b, Crhr2, D0H4S114, Ddit3,Deb1, Dexi, Dhrs7c, Eif4e, Endog, Epha7, Exd2, Fam69a, Fhod3, Fn3k,Fndc5, Fsd2, Gcom1, Gdap1, Gm4841, Gm5105, Gm9909, Gnb5, Gpd2, Grtp1,Heatr5a, Hlf, Homer1, Ikzf2, Inppl1, Irx3, Itgb6, Jarid2, Jph2, Khdrbs3,Klf7, Klhl23, Ky, Lrp2 bp, Lrrfip1, Map2k6, Map3k4, Mat2a, Mkks, Mkl1,Mrc2, Mreg, Mrpl39, Narf, Ntf5, Nudt3, Osbpl6, Ostc, Parp8, Pkia, Plcd4,Podxl, Polk, Polr3k, Ppm1l, Pppde2, Prss23, Psd3, Psph, Ptpmt1, Ptx3,Qrsl1, Rasgrp3, Rhobtb3, Ric8b, Rnf150, Rsph1, Rundc1, Rxrg, Sel1l3,Sema3a, Sgcd, Shisa2, Sirt5, Slc25a19, Slc41a3, Slc4a4, Slco5a1,Snrnp35, Stac3, Ston2, Stradb, Stxbp4, Tfrc, Tmc7, Tmem218, Tmtc1,Tnfaip2, Tob1, Trim35, Ttl, Vegfa, and Vgll4.

24. Muscle Atrophy Signature-4

Induced and repressed mRNA were evaluated for muscle atrophysignature-4. The statisitical significance for the identified mRNAs wasdefined as P≤0.01.

For induced mRNAs: mouse tibialis anterior mRNAs significantly inducedby 1 week of denervation and significantly induced by 1 week of Gadd45aoverexpression.

For repressed mRNAs: mouse tibialis anterior mRNAs significantlyrepressed by 1 week of denervation and significantly repressed by 1 weekof Gadd45a overexpression.

The identified induced mRNAs included 2410089E03Rik, 6720456H20Rik,Abca1, Abhd2, Abr, Aifl1, Akap6, Alg8, Alox5ap, mpd3, Ankrd1, Anxa4,Aoah, App, Araf, Arfgap3, Arhgef2, Arpc3, Arpp21, Atf7ip, Atp6ap2,Atp6v1h, Atp7a, Atp8b1, B4galt5, Bax, Baz2a, Bhlhb9, Bmp2k, C3ar1, Canx,Casp3, Ccdc111, Ccdc122, Ccdc93, Ccndbp1, Cct4, Cd68, Cd82, Cdkn1a,Cep192, Cgref1, Chd4, Chrna1, Chrnb1, Chrng, Chuk, Clec12a, Clec4a3,Col19a1, Copb2, Cpne2, Cstb, Ctnna1, Ctps2, Ctsd, Ctsl, Ctss, Ctsz,Cyb5r3, Cybb, Cyr61, D10Wsu52e, D930016D06Rik, Dcaf13, Dclre1c, Dctn5,Ddb1, Ddhd1, Decr2, Derl1, Dhx9, Dido1, Dnajc1, Eda2r, Eef1b2, Eef2,Emr1, Epb4.1l3, Erbb2ipm, Erlin1, Esyt1, Fam108c, Fam115a, Fbxo30,Frrs1, Fst, Fubp1, Fyb, Gab2, Gabarap, Gadd45a, Galec, Galnt7, Ganab,Gigyf2, Gm3435, Gnb2l1, Gng2, Gnl2, Gnl3, Gprasp1, Gpsm2, Gramd1b, H19,H2-Aa, Hmgn3, Hn1, Hnrnpu, Hprt, Hsp90ab1, Hsp90b1, Hspa2, Hspa4, Hspb8,Htatip2, Id2, Ifi30, Igbp1, Igdcc4, Ilf3, Imp4, Impact, Irak4, Itm2b,Ivns1abp, Kcnn3, Kdm3a, Khdrbs1, Kif5b, Klhl5, Krt18, Lbh, Lgals3, Lgmn,Lpar6, Lpin2, Lyz2, Macf1, Map1lc3a, Map3k1, Map4k4, Marveld2, Matr3,Mcm6, Mdm2, Mdm4, Me2, Med12, Mgea5, Micall1, Mpp1, Mrc1, Mtap1b, Myf6,Myl4, Myo5a, Ncam1, Nip7, Nln, Nop58, Nrcam, Nup93, Nvl, Obfc2a, Osbpl8,Palm2, Parp4, Pcbd1, Pcgf3, Pdlim3, Pfn1, Pgd, Pik3r3, Plaa, Plekha5,Plxdc2, Plxna1, Polr2a, Polr3b, Ppfibp1, Ppib, Prep, Prkdc, Prmt1,Prss48, Prune2, Psmb1, Psmd5, Rad50, Rassf4, Rb1, Rbm45, Reep5, Rgs2,Riok3, Rlim, Rnasel, Rpl31, Rps3, Rps9, Rrad, Rras2, Rspry1, Runx1,Sap30 bp, Sema4d, Sema6a, Serf1, Serpinb6a, Sesn3, Sf3b1, Sf3b3, Sgpl1,Sh3d19, Sh3pxd2a, Sh3rf1, Sik1, Sirpa, Slc20a1, Slc25a24, Slc9a7,Slc9a9, Sin, Smarcad1, Smc1a, Smc5, Snd1, Snx5, Spin1, Srp14, Ssu72,Stam, Supt5h, Tbcld8, Tbcd, Tbxas1, Tec, Tgfbr1, Tgs1, Thoc5, Thumpd3,Tiam2, Tlr4, Tlr6, Tmeff1, Tmem176b, Tmem179b, Tmem209, Tmem38b, Tnc,Tnfrsf22, Tnfrsf23, Tnnt2, Trim25, Trp63, Tubb5, Tubb6, Tyrobp, Uchl1,Ugcg, Usp11, Usp5, Wasf2, Wbp5, Wbscr27, Wdr36, Wdr61, Wdr67, Wdr77,Wdyhv1, Wsb1, Ylpm1, Ypel2, Ywhab, Zfp280d, Zfp318, Zfp346, Zfp36l1, andZmynd8.

The identified repressed mRNAs included 0610012G03Rik, 1110001J03Rik,1110067D22Rik, 2010106G01Rik, 2310002L09Rik, 2310003L22Rik,2310010M20Rik, 2610507B11Rik, 2610528E23Rik, 2810407C02Rik,4931409K22Rik, 4933403F05Rik, 5730437N04Rik, 9630033F20Rik, A2ld1,A930018M24Rik, Abcb1a, Abcb4, Abcd2, Abi3 bp, Acaa2, Acadm, Acadv1,Acat1, Acot13, Adal, Adcy10, Adk, Adssl1, Aes, AI317395, Aimp2, Ak1,Alas2, Aldh1a1, Ank, Ank1, Ankrd9, Ano2, Ano5, Aplp2, Apobec2, Aqp4, Ar,Arhgap19, Arhgap20, Arhgap31, Arl3, Asb10, Asb11, Asb12, Asb14, Asb15,Atp11a, Atp13a5, Atp1b1, Atp5a1, Atp5e, Atp8a1, Atxn1, B4galt4, Bckdk,Bhlhe41, Bpgm, Bpil1, Brp44, Btbd1, C2cd2, Camk2a, Camk2g, Capn5, Car8,Cast, Cc2d2a, Ccng1, Ccnk mCd34, Cd36, Cdc14a, Cdc42ep3, Cdh5, Cdnf,Ces1d, Chchd10, Chchd3, Cib2, Ckm, Clcn1, Clic5, Cmbl, Cntfr, Col11a1,Coq9, Cox11, Cox6a2, Cox8b, Cpt1b, Csrp2 bp, Cuedc1, Cyb5b, Cyyr1,D0H4S114, D1Ertd622e, Dab2ip, Dcun1d2, Deb1, Decr1, Dgkb, Dhrs7c, Dlat,Dlc1d, Dlg1, Dlst, Dnajb5, Dusp28, Ecsit, Eef1a2, Eepd1, Efcab2, Eif4e,Endog, Eno3, Epas1, Epha7, Etfb, Exd2, Eya1, Fam132a, Fastkd3, Fbp2,Fbxo3, Fdx1, Fez2, Fgfbp1, Fh1, Fitm2, Flt1, Fmo5, Fsd2, Fxyd1, Fzd4,G3bp2, Ganc, Gbas, Gcom1, Gdap1, Ghr, Gjc3, Glb1l2, Gm4841, Gm4861,Gm4951, Gm5105, Gmpr, Gpcpd1, Gpd1, Gpd2, Gpt2, Grsf1, Gucy1a3, Gys1,Hadh, Hfe2, Hivep2, Hk2, Hlf, Homer1, Hsdl2, Idh3a, Idh3g, Il15ra,Inpp5a, Irx3, Jak2, Jam2, Jph1, Kcna7, Kcnj2, Kcnn2, Kdr, Khdrbs3,Kif1b, Kif1c, Kit1, Klf12, Klhl23, Klhl31, Klhl31, Klhl7, Ky, Ldb3,Lifr, Lmbr1, Lphn1, Lpin1, Lpl, Lrig1, Lrrc39, Lynx1, Man2a2, Maob,Map2k6, Map2k7, Map3k4, Mapkapk2, Mbn1, Mccc1, Mdh1, Mdh2, Me3, Mfn1,Mfn2, Mgst3, Mlf1, Mpnd, Mpz, Mr1, Mreg, Mtus1, Mybpc2, Myo5c, Myom2,Myoz1, Narf, Ndrg2, Ndufa3, Ndufa5, Ndufa8, Ndufb8, Ndufb9, Ndufs1,Ndufs2, Ndufs6, Ndufs8, Ndufv1, Nf2, Nos1, Nr1d1, Nudt3, Oat, Ociad2,Ocrl, Osbpl6, Osgepl1, Ostn, Paqr9, Parp3, Pcmtd1, Pcnt, Pcnx, Pdgfd,Pdha1, Pdpr, Pfkfb3, Pfkm, Pfn2, Pgam2, Phb, Phka1, Phkg1, Phtf2, Phyh,Pitpnc1, Pkdcc, Pkia, Pla2g12a, Pla2g4e, Plcb1, Plcd4, Pln, Pmp22,Ppara, Ppargc1a, Ppat, Ppm1a, Ppml1, Ppp1cb, Ppp1r1a, Ppp2r2a, Ppp3cb,Prelp, Prkab2, Prkca, Prkg1, Ptp4a3, Ptprb, Pttg1, Pxmp2, Pygm, Rab28,Rasgrp3, Rcan2, Rgs5, Rhot2, Rnf123, Rpa1, Rpl31, Rtn4ip1, Samd12,Samd5, Satb1, Scn4a, Scn4b, Sdha, Sdhb, Sdr39u1, Sel1l3, Sema6c,Serpine2, Shisa2, Slc15a5, Slc16a3, Slc19a2, Slc24a2, Slc25a11,Slc25a12, Slc25a3, Slc25a4, Slc2a12, Slc2a4, Slc35f1, Slc37a4, Slc43a3,Slc4a4, Slc6a13, Slc6a8, Slc9a3r2, Slco3a1, Smarca1, Smox, Smyd1, Snrk,Sorbs2, Spop, Srl, St3gal3, St3gal6, St6galnac6, Stk381, Stradb, Strbp,Strbp, Stxbp4, Suclg1, Tab2, Taf3, Tarsl2, Tcea3, Thra, Tiam1, Timp4,Tln2, Tmem126a, Tmem126b, Tmem65, Tnfaip2, Tnmd, Tnnc2, Tnni2, Tnxb,Tomm40l, Trak1, Trak2, Trim24, Trpc3, Tuba8, Txlnb, Txnip, U05342, Uaca,Ulk2, Uqcrc1, Uqcrfs1, Uqcrq, Vamp5, Vdac1, Vegfa, Vegfb, Xpr1, Yipf7,Zfand5, Zfp191, and Zfp238.

F. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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The invention claimed is:
 1. A method for: (a) increasing skeletalmuscle mass; (b) reducing skeletal muscle atrophy; (c) increasingmuscular strength; (d) promoting muscle growth; (e) decreasing musclewasting; or (f) increasing strength per unit of muscle mass in an animalidentified or having been identified to be in need of one or more of(a)-(f), the method comprising administering to the animal an effectiveamount of a compound of formula:

or a stereoisomer, tautomer, solvate, or pharmaceutically acceptablesalt thereof, thereby accomplishing one or more of (a)-(f), wherein theanimal is not a human or a rabbit.
 2. The method according to claim 1,for increasing skeletal muscle mass, wherein the compound is

or a stereoisomer or pharmaceutically acceptable salt thereof.
 3. Themethod according to claim 1, for reducing skeletal muscle atrophy,wherein the compound is

or a stereoisomer or pharmaceutically acceptable salt thereof.
 4. Themethod according to claim 1, for increasing muscular strength, whereinthe compound is

or a stereoisomer or pharmaceutically acceptable salt thereof.
 5. Themethod according to claim 1, for promoting muscle growth, wherein thecompound is

or a stereoisomer or pharmaceutically acceptable salt thereof.
 6. Themethod according to claim 1, for decreasing muscle wasting, wherein thecompound is:

or a stereoisomer or pharmaceutically acceptable salt thereof.
 7. Themethod according to claim 1, for increasing strength per unit of musclemass, wherein the compound is

or a stereoisomer or pharmaceutically acceptable salt thereof.
 8. Themethod according to claim 1, comprising administering to the animalgreater than 75 mg per day of the compound or stereoisomer, tautomer,solvate, or pharmaceutically acceptable salt thereof.
 9. The methodaccording to claim 1, comprising administering to the animal greaterthan 100 mg per day of the compound or stereoisomer, tautomer, solvate,or pharmaceutically acceptable salt thereof.
 10. The method according toclaim 1, wherein the compound is

or a stereoisomer or pharmaceutically acceptable salt thereof.
 11. Themethod according to claim 1, comprising administering to the animalgreater than 150 mg per day of the compound or stereoisomer, tautomer,solvate, or pharmaceutically acceptable salt thereof.
 12. The methodaccording to claim 11, wherein the compound is

or a stereoisomer or pharmaceutically acceptable salt thereof.
 13. Themethod according to claim 1, comprising administering to the animal atleast 5.0 mg/kg per day of the compound or stereoisomer, tautomer,solvate, or pharmaceutically acceptable salt thereof.
 14. The methodaccording to claim 13, wherein the compound is

or a stereoisomer, or pharmaceutically acceptable salt thereof.
 15. Themethod according to claim 1, comprising administering to the animal 0.5to 250 mg/kg per day of the compound or stereoisomer, tautomer, solvate,or pharmaceutically acceptable salt thereof.
 16. The method according toclaim 15, wherein the compound is

or a stereoisomer or pharmaceutically acceptable salt thereof.
 17. Themethod according to claim 1, wherein said animal is selected from thegroup consisting of a non-human primate, domesticated fish, domesticatedcrustacean, domesticated mollusk, poultry, dog, cat, and livestock. 18.The method according to claim 17, wherein the compound is

or a stereoisomer or pharmaceutically acceptable salt thereof.
 19. Themethod according to claim 1, wherein said animal is selected from thegroup consisting of a non-human primate, horse, pig, dog, sheep, goat,cow, cat, guinea pig, fish, and bird.
 20. The method according to claim19, wherein the compound is

or a stereoisomer or pharmaceutically acceptable salt thereof.